Cancer immunotherapy by disrupting pd-1/pd-l1 signaling

ABSTRACT

The disclosure provides a method for immunotherapy of a cancer patient, comprises administering to the patient an Ab that inhibits signaling from the PD-1/PD-L1 signaling pathway, or a combination of such Ab and an anti-CTLA-4 Ab. This disclosure also provides a method for immunotherapy of a cancer patient comprising selecting a patient who is a suitable candidate for immunotherapy based on an assessment that the proportion of cells in a test tissue sample from the patient that express PD-L1 on the cell surface exceeds a predetermined threshold level, and administering an anti-PD-1 Ab to the selected subject. The disclosure additionally provides rabbit mAbs that bind specifically to a cell surface-expressed PD-L1 antigen in a FFPE tissue sample, and an automated IHC method for assessing cell surface expression in FFPE tissues using the provided anti-PD-L1 Abs.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/647,442, filed May 15, 2012, and U.S. ProvisionalPatent Application No. 61/790,747, filed Mar. 15, 2013, the contents ofwhich are hereby incorporated herein by reference in their entirety.

Throughout this application, various other publications are referencedin parentheses by author name and date, or by patent No. or patentPublication No. Full citations for these publications may be found atthe end of the specification immediately preceding the claims. Thedisclosures of these publications are hereby incorporated in theirentireties by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein. However, thecitation of a reference herein should not be construed as anacknowledgement that such reference is prior art to the presentinvention.

FIELD OF THE INVENTION

This invention relates to methods for immunotherapy of a cancer patientcomprising administering to the patient antibodies that disrupt thePD-1/PD-L1 signaling pathway. A biomarker may be used as part of thistreatment for identifying suitable patients for immunotherapy and forpredicting the efficacy of anti-PD-1 treatment.

BACKGROUND OF THE INVENTION

Human cancers harbor numerous genetic and epigenetic alterations,generating neoantigens potentially recognizable by the immune system(Sjoblom et al., 2006). The adaptive immune system, comprised of T and Blymphocytes, has powerful anti-cancer potential, with a broad capacityand exquisite specificity to respond to diverse tumor antigens. Further,the immune system demonstrates considerable plasticity and a memorycomponent. The successful harnessing of all these attributes of theadaptive immune system would make immunotherapy unique among all cancertreatment modalities. However, although an endogenous immune response tocancer is observed in preclinical models and patients, this response isineffective, and established cancers are viewed as “self” and toleratedby the immune system. Contributing to this state of tolerance, tumorsmay exploit several distinct mechanisms to actively subvert anti-tumorimmunity. These mechanisms include dysfunctional T-cell signaling(Mizoguchi et al., 1992), suppressive regulatory cells (Facciabene etal., 2012), and the co-opting of endogenous “immune checkpoints,” whichserve to down-modulate the intensity of adaptive immune responses andprotect normal tissues from collateral damage, by tumors to evade immunedestruction (Topalian et al., 2011; Mellman et al., 2011).

Until recently, cancer immunotherapy had focused substantial effort onapproaches that enhance anti-tumor immune responses by adoptive-transferof activated effector cells, immunization against relevant antigens, orproviding non-specific immune-stimulatory agents such as cytokines. Inthe past decade, however, intensive efforts to develop specific immunecheckpoint pathway inhibitors have begun to provide newimmunotherapeutic approaches for treating cancer, including thedevelopment of an antibody (Ab), ipilimumab (YERVOY®), that binds to andinhibits Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) for the treatment ofpatients with advanced melanoma (Hodi et al., 2010) and, as describedherein, the development of Abs that block the inhibitory PD-1 pathway.

Programmed Death-1 (PD-1) is a key immune checkpoint receptor expressedby activated T and B cells and mediates immunosuppression. PD-1 is amember of the CD28 family of receptors, which includes CD28, CTLA-4,ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1have been identified, Programmed Death Ligand-1 (PD-L1) and ProgrammedDeath Ligand-2 (PD-L2), that are expressed on antigen-presenting cellsas well as many human cancers and have been shown to downregulate T cellactivation and cytokine secretion upon binding to PD-1 (Freeman et al.,2000; Latchman et al., 2001). Unlike CTLA-4, PD-1 primarily functions inperipheral tissues where activated T-cells may encounter theimmunosuppressive PD-L1 (B7-H1) and PD-L2 (B7-DC) ligands expressed bytumor and/or stromal cells (Flies et al., 2011; Topalian et al., 2012a).Inhibition of the PD-1/PD-L1 interaction mediates potent antitumoractivity in preclinical models (U.S. Pat. Nos. 8,008,449 and 7,943,743),and the use of Ab inhibitors of the PD-1/PD-L1 interaction for treatingcancer has entered clinical trials (Brahmer et al., 2010; Topalian etal., 2012b; Brahmer et al., 2012; Flies et al., 2011; Pardoll, 2012;Hamid and Carvajal, 2013).

The promise of the emerging field of personalized medicine is thatadvances in pharmacogenomics will increasing be used to tailortherapeutics to defined sub-populations, and ultimately, individualpatients in order to enhance efficacy and minimize adverse effects.Recent successes include, for example, the development of imatinibmesylate (GLEEVEC®), a protein tyrosine kinase inhibitor that inhibitsthe bcr-abl tyrosine kinase, to treat Philadelphia chromosome-positivechronic myelogenous leukemia (CML); crizotinib (XALKORI®) to treat the5% of patients with late-stage non-small cell lung cancers who express amutant anaplastic lymphoma kinase (ALK) gene; and vemurafenib(ZELBORAF®), an inhibitor of mutated B-RAF protein (V600E-BRAF) which isexpressed in around half of melanoma tumors. However, unlike theclinical development of small molecule agents that target discreteactivating mutations found in select cancer populations, a particularchallenge in cancer immunotherapy has been the identification ofmechanism-based predictive biomarkers to enable patient selection andguide on-treatment management. Advances in validating PD-L1 expressionas a biomarker for screening patients for anti-PD-1 immunotherapy aredescribed herein.

SUMMARY OF THE INVENTION

The present disclosure provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises administering to thesubject a composition comprising a therapeutically effective amount ofan agent that disrupts, reduces or suppresses signaling from aninhibitory immunoregulator. In preferred embodiments, the agent is anAb. In other preferred embodiments, the inhibitory immunoregulator is acomponent of the PD-1/PD-L1 signaling pathway. In further preferredembodiments, the Ab disrupts the interaction between PD-1 and PD-L1. Incertain embodiments, the Ab is an anti-PD-1 Ab of the invention or ananti-PD-L1 Ab of the invention. In preferred embodiments, the anti-PD-1Ab of the invention is nivolumab (BMS-936558) and the anti-PD-L1 Ab ofthe invention is BMS-936559. In certain embodiments, the subject hasbeen pre-treated for the cancer. In other embodiments, the cancer is anadvanced, metastatic and/or refractory cancer. In preferred embodiments,the administration of the Ab or antigen-binding portion to the subjectthereof induces a durable clinical response in the subject.

This disclosure also provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is a suitable candidate for immunotherapy, the selecting comprising(i) optionally providing a test tissue sample obtained from a patientwith cancer of the tissue, the test tissue sample comprising tumor cellsand tumor-infiltrating inflammatory cells, (ii) assessing the proportionof cells in the test tissue sample that express PD-L1 on the cellsurface, and (iii) selecting the subject as a suitable candidate basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface exceeds a predetermined thresholdlevel; and (b) administering a composition comprising a therapeuticallyeffective amount of an agent that disrupts signaling from the PD-1/PD-L1pathway, for example, an anti-PD-1 or an anti-PD-L1 Ab, to the selectedsubject.

The disclosure further provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is not suitable for immunotherapy with an agent that disruptssignaling from the PD-1/PD-L1 pathway, for example, an anti-PD-1 or ananti-PD-L1 Ab, the selecting comprising (i) optionally providing a testtissue sample obtained from a patient with cancer of the tissue, thetest tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells; (ii) assessing the proportion of cells in the testtissue sample that express PD-L1 on the cell surface; and (iii)selecting the subject as not suitable for said immunotherapy based on anassessment that the proportion of cells in the test tissue sample thatexpress PD-L1 on the cell surface is less than a predetermined thresholdlevel; and (b) administering a standard-of-care therapeutic other thanan agent that disrupts signaling from the PD-1/PD-L1 pathway to theselected subject.

In addition, the disclosure provides a method for selecting a cancerpatient for immunotherapy with an agent that disrupts signaling from thePD-1/PD-L1 pathway, for example, an anti-PD-1 or an anti-PD-L1 Ab, whichmethod comprises: (a) optionally providing a test tissue sample obtainedfrom a patient with cancer of the tissue, the test tissue samplecomprising tumor cells and tumor-infiltrating inflammatory cells; (b)assaying the test tissue sample to determine the proportion of cellstherein that express PD-L1 on the cell surface; (c) comparing theproportion of cells that express PD-L1 on the cell surface with apredetermined threshold proportion; and (d) selecting the patient forimmunotherapy based on an assessment that PD-L1 is expressed in cells ofthe test tissue sample.

This disclosure further provides a method for predicting the therapeuticeffectiveness of an agent that disrupts signaling from the PD-1/PD-L1pathway, for example, an anti-PD-1 or an anti-PD-L1 Ab, for treating acancer patient, which method comprises: (a) optionally providing a testtissue sample obtained from a patient with cancer of the tissue, thetest tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells; (b) assaying the test tissue sample to determine theproportion of cells therein that express PD-L1 on the cell surface; (c)comparing the proportion of cells that express PD-L1 on the cell surfacewith a predetermined threshold value; and (d) predicting the therapeuticeffectiveness of the agent, wherein if the proportion of cells thatexpress PD-L1 on the cell surface exceeds the threshold proportion theagent is predicted to be effective in treating the patient, and whereinif the proportion of cells that express PD-L1 on the cell surface isbelow the threshold proportion the agent is predicted to not beeffective in treating the patient.

The present disclosure also provides a method for determining animmunotherapeutic regimen comprising an agent that disrupts signalingfrom the PD-1/PD-L1 pathway, for example, an anti-PD-1 or an anti-PD-L1Ab, for treating a cancer patient, which method comprises: (a)optionally providing a test tissue sample obtained from a patient withcancer of the tissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (b) assaying the test tissuesample to determine the proportion of cells therein that express PD-L1on the cell surface; (c) comparing the proportion of cells that expressPD-L1 on the cell surface with a predetermined threshold proportion; and(d) determining an immunotherapeutic regimen comprising the agent basedon the determination that the proportion of cells that express PD-L1 onthe cell surface exceeds the predetermined threshold proportion.

In certain embodiments of the methods described herein, the test tissuesample is a formalin-fixed and paraffin-embedded (FFPE) sample. Incertain other embodiments, assessing the proportion of cells in the testtissue sample that express PD-L1 on the cell surface is achieved byimmunohistochemical (IHC) staining of the FFPE sample. In preferredembodiments, the mAb 28-8 or 5H1 is used in an automated IHC assay tobind to PD-L1 on the surface of cells in the test tissue sample. Inpreferred embodiments of any of the methods disclosed herein, the canceris melanoma (MEL), renal cell carcinoma (RCC), squamous non-small celllung cancer (NSCLC), non-squamous NSCLC, colorectal cancer (CRC),castration-resistant prostate cancer (CRPC), hepatocellular carcinoma(HCC), squamous cell carcinoma of the head and neck, carcinomas of theesophagus, ovary, gastrointestinal tract and breast, or a hematologicmalignancy such as multiple myeloma, B-cell lymphoma, T-cell lymphoma,Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, and chronicmyelogenous leukemia.

This invention additionally provides a mAb or antigen-binding portionthereof that binds specifically to a cell surface-expressed human PD-L1antigen in a FFPE tissue sample. In preferred embodiments, the mAb orantigen-binding portion thereof does not bind to a cytoplasmic PD-L1antigen in the FFPE tissue sample. In other preferred embodiments, themonoclonal Ab (mAb) is the rabbit mAb designated 28-8, 28-1, 28-12, 29-8or 20-12, or the mouse mAb designated 5H1.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all cited references, includingscientific articles, newspaper reports, GenBank entries, patents andpatent applications cited throughout this application are expresslyincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Cross-competition between 5C4 and other HuMab anti-PD-1mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, the5C4 Fab fragment substantially blocked the binding of mAbs 5C4 itself,as well as the binding of 2D3 and 7D3; B, the 5C4 Fab fragmentsubstantially blocked the binding of mAb 4H1; C, the 5C4 mAbsubstantially blocked the binding of mAb 17D8.

FIGS. 2A-2F. Cross-competition of FITC-conjugated human anti-hPD-L1 mAbsfor binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, Bindingof labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and wassignificantly blocked by itself; B, Binding of labeled 3G10 wassignificantly blocked by each of the tested anti-PD-L1 Abs except 10H10;C, Binding of labeled 10A5 was significantly blocked by each of thetested anti-PD-L1 Abs except 10H10; D, Binding of labeled 11E6 wassignificantly blocked by each of the tested anti-PD-L1 Abs except 10H10;and E, Binding of labeled 12A4 was significantly blocked by each of thetested anti-PD-L1 Abs except 10H10; and F, Binding of labeled 13G4 wassignificantly blocked by each of the tested anti-PD-L1 Abs except 10H10.

FIG. 3. Cross-competitive inhibition of binding of biotinylated mAb 12A4to ES-2 cells by human anti-hPD-L1 mAbs. Fluorescence of boundbiotin-12A4 is plotted against the concentration of unlabeled hPD-L1HuMabs.

FIG. 4. Spider plot showing activity of anti-PD-1 mAb in patients withtreatment-refractory melanoma (MEL). A representative plot of changes intumor burden over time demonstrates the time course of change in the sumof the longest diameters of target lesions, compared with baseline, in27 MEL patients treated with 5C4 at a dose of 1.0 mg/kg. In the majorityof patients who achieved an objective response (OR), responses weredurable and evident by the end of cycle 3 (6 months) of treatment(vertical dashed line). Tumor regressions followed conventional as wellas “immune-related” patterns of response, such as prolonged reduction intumor burden in the presence of new lesions.

FIG. 5. Activity of anti-PD-1 mAb in patient with metastatic RCC.Partial regression of metastatic RCC in a 57-year-old patient treatedwith 5C4 at 1 mg/kg is illustrated. This patient had previouslyundergone radical surgery and had developed progressive disease afterreceiving sunitinib, temsirolimus, sorafenib, and pazopanib. Arrows showregression of recurrent tumor in the operative field.

FIG. 6. Activity of anti-PD-1 mAb in patient with metastatic MEL. Acomplete response of metastatic MEL is illustrated in a 62-year-oldpatient treated with 5C4 at 3 mg/kg, associated with vitiligo. (i)Pretreatment CT scan, inguinal lymph node metastasis (arrow); (ii) after13 months of treatment. Numerous metastases in the subcutaneous tissueand retroperitoneum also regressed completely (not shown). Vitiligodeveloped after 6 months of treatment; photos taken at 9 months undervisible light (iii) and ultraviolet light (iv). Skin biopsies withimmunohistochemistry for microphthalmia-associated transcription factor(MITF) show melanocytes (arrows) at the epidermal-dermal junction innormal skin (v), and scarce (vi) or absent (vii) melanocytes in skinpartially or fully affected by vitiligo.

FIG. 7. Activity of anti-PD-1 mAb in patient with metastatic NSCLC. Apartial response is illustrated in a patient with metastatic NSCLC(nonsquamous histology) treated with 5C4 at 10 mg/kg. Arrows showinitial progression in pulmonary lesions followed by regression(“immune-related” pattern of response).

FIGS. 8A and 8B. Correlation between tumor PD-L1 expression andanti-PD-1 clinical response. Pretreatment tumor cell surface expressionof PD-L1, as determined by IHC on formalin-fixed paraffin-embeddedspecimens, correlates with OR to PD-1 blockade. Forty-two subjects withadvanced cancers including melanoma, non-small cell lung cancer,colorectal cancer, renal cell cancer, and castration-resistant prostatecancer (n=18, 10, 7, 5, and 2, respectively) were studied. A, There wasa significant correlation of tumor cell surface PD-L1 expression withobjective clinical response. No patients with PD-L1 negative tumorsexperienced an OR. B, Examples of IHC analysis with the anti-PD-L1 mAb5H1 are shown in a melanoma lymph node metastasis (top), a renal cellcancer nephrectomy specimen (middle), and a lung adenocarcinoma brainmetastasis (bottom). All 400× original magnification. Arrows indicateone of many tumor cells in each specimen with surface membrane stainingfor PD-L1. Asterisk indicates a normal glomerulus in the nephrectomyspecimen, which is negative for PD-L1 staining.

FIG. 9. Graphical comparison of the binding of mAbs 28-8 and 5H1 toPD-L1 antigen in tumor tissues by histoscore analysis. The rabbit mAb28-8 showed higher histoscores in 7 out of 10 samples tested.

FIGS. 10A-10D. Spider plot showing activity of anti-PD-L1 mAb inpatients with treatment-refractory MEL and NSCLC. Representative plotsdemonstrate the time course of target lesion tumor burden over time inpatients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10mg/kg (C) and in patients with NSCLC treated at 10 mg/kg (D). In themajority of patients who achieved ORs, responses were durable and wereevident by the end of cycle 2 (3 months) of treatment, irrespective ofdose or tumor type. Tumor regressions followed conventional as well as“immune-related” patterns of response.

FIG. 11. Complete response in a patient with melanoma treated withBMS-936559 at 3 mg/kg. Circles indicate an initial increase in the sizeof pulmonary nodules at 6 weeks and 3 months followed by completeregression at 10 months (“immune-related” pattern of response).

FIG. 12. Complete response in a patient with melanoma treated withBMS-936559 at 1 mg/kg. This patient developed an isolated brainmetastasis 3 months after initiation of treatment that was successfullytreated with stereotactic radiosurgery. A partial response in abdominaldisease (circled) was noted at 8 months, with no evidence of disease at15 months.

FIG. 13. Partial response in a patient with NSCLC (non-squamoushistology) treated with BMS-936559 at 10 mg/kg. Note the response indisease in right lung pleura and liver.

FIGS. 14A and 14B. Clinical activity of MEL patients who received aconcurrent regimen of nivolumab and ipilimumab. A, Representative spiderplots show changes from baseline in the tumor burden, measured as thesum of products of perpendicular diameters of all target lesions, inpatients who received the concurrent regimen of 1 mg/kg nivolumab+3mg/kg ipilimumab, the MTD. Triangles indicate the first occurrence of anew lesion. B, A representative waterfall plot shows maximum percentageresponse in baseline target lesions in patients who received theconcurrent regimen.

FIG. 15. Tumor regressions of a 52-year old MEL patient who received aconcurrent regimen of 1 mg/kg nivolumab+3 mg/kg ipilimumab. This patientpresented with extensive neck, mediastinal, axillary, abdominal andpelvic lymphadenopathy, bilateral pulmonary nodules, small bowelmetastasis, peritoneal implants and diffuse subcutaneous nodules.Baseline lactate dehydrogenase (LDH) was 2.25× upper limit of normal,hemoglobin was 9.7 g/dL, and symptoms included nausea and vomiting.Within 4 weeks of treatment the LDH normalized, symptoms improved(appetite increased, nausea decreased), and cutaneous lesions wereregressing. At week-12 scans, there was marked reduction in all areas ofdisease. Arrows denote location of metastatic disease.

FIGS. 16A-16C. Clinical activity of MEL patients who received variousconcurrent regimens of nivolumab and ipilimumab. Representative spiderplots show changes from baseline in the tumor burden, measured as thesum of products of perpendicular diameters of all target lesions, inpatients who received a concurrent regimen of 0.3 mg/kg nivolumab+3mg/kg ipilimumab (A), 3 mg/kg nivolumab+1 mg/kg ipilimumab (B), or 3mg/kg nivolumab+3 mg/kg ipilimumab (C). Triangles indicate the firstoccurrence of a new lesion.

FIG. 17. Tumor regressions of a 61-year old MEL patient who received aconcurrent regimen of 0.3 mg/kg nivolumab+3 mg/kg ipilimumab. Thepatient presented with stage IV (M1c) MEL of an unknown primary site,metastatic to the stomach and the mesentery. Lactate dehydrogenase was225 and hemoglobin was 9.6 g/dL after a recent transfusion. Twelve weeksafter the initiation of study treatment, a CT scan showed an 86%reduction in the bulky disease burden.

FIGS. 18A-18C. Clinical activity of MEL patients who received asequenced regimen of nivolumab and ipilimumab. Representative spiderplots show changes from baseline in the tumor burden, measured as thesum of products of perpendicular diameters of all target lesions, inpatients who received a sequenced regimen of 1 mg/kg nivolumab (A) or 3mg/kg nivolumab (B) after prior ipilimumab therapy. Triangles indicatethe first occurrence of a new lesion. C, A representative waterfall plotshows maximum percentage response in baseline target lesions in patientswho received a sequenced regimen; “*” denotes patients who hadradiographic progression with prior ipilimumab treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for immunotherapy of a subjectafflicted with diseases such as cancer or an infectious disease, whichmethods comprise administering to the subject a composition comprising atherapeutically effective amount of a compound or agent that potentiatesan endogenous immune response, either stimulating the activation of theendogenous response or inhibiting the suppression of the endogenousresponse. More specifically, this disclosure provides methods forpotentiating an endogenous immune response in a subject afflicted withcancer so as to thereby treat the patient, which method comprisesadministering to the subject a therapeutically effective amount of anagent, such as an Ab or an antigen-binding portion thereof, thatdisrupts or inhibits signaling from an inhibitory immunoregulator. Incertain embodiments, the inhibitory immunoregulator is a component ofthe PD-1/PD-L1 signaling pathway. Accordingly, certain embodiments ofthe invention provide methods for immunotherapy of a subject afflictedwith cancer, which methods comprise administering to the subject atherapeutically effective amount of an Ab or an antigen-binding portionthereof that disrupts the interaction between the PD-1 receptor and itsligand, PD-L1. In certain preferred embodiments, the Ab orantigen-binding portion thereof binds specifically to PD-1. In otherpreferred embodiments, the Ab or antigen-binding portion thereof bindsspecifically to PD-L1. Certain embodiments comprise the use of ananti-PD-1 Ab is in combination with another anti-cancer agent,preferably an anti-CTLA-4 Ab, to treat cancer. In certain otherembodiments, the subject is selected as suitable for immunotherapy in amethod comprising measuring the surface expression of PD-L1 in a testtissue sample obtained from a patient with cancer of the tissue, forexample, determining the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface, and selecting the patient forimmunotherapy based on an assessment that PD-L1 is expressed on thesurface of cells in the test tissue sample.

TERMS

In order that the present disclosure may be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

“Administering” refers to the physical introduction of a compositioncomprising a therapeutic agent to a subject, using any of the variousmethods and delivery systems known to those skilled in the art.Preferred routes of administration for Abs of the invention includeintravenous, intramuscular, subcutaneous, intraperitoneal, spinal orother parenteral routes of administration, for example by injection orinfusion. The phrase “parenteral administration” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intralymphatic,intralesional, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an Ab of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. For example, an adverse event may be associated withactivation of the immune system or expansion of immune system cells(e.g., T cells) in response to a treatment. A medical treatment may haveone or more associated AEs and each AE may have the same or differentlevel of severity. Reference to methods capable of “altering adverseevents” means a treatment regime that decreases the incidence and/orseverity of one or more AEs associated with the use of a differenttreatment regime.

An “antibody” (Ab) shall include, without limitation, a glycoproteinimmunoglobulin which binds specifically to an antigen and comprises atleast two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds, or an antigen-binding portion thereof. Each H chaincomprises a heavy chain variable region (abbreviated herein as V_(H))and a heavy chain constant region. The heavy chain constant regioncomprises three constant domains, C_(H1), C_(H2) and C_(H3). Each lightchain comprises a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion is comprises one constant domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the Abs may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁵ to10⁻¹¹ M⁻¹ or less. Any K_(D) greater than about 10⁻⁴ M⁻¹ is generallyconsidered to indicate nonspecific binding. As used herein, an Ab that“binds specifically” to an antigen refers to an Ab that binds to theantigen and substantially identical antigens with high affinity, whichmeans having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less, evenmore preferably 5×10⁻⁹ M or less, and most preferably between 10⁻⁸ M and10⁻¹⁰ M or less, but does not bind with high affinity to unrelatedantigens. An antigen is “substantially identical” to a given antigen ifit exhibits a high degree of sequence identity to the given antigen, forexample, if it exhibits at least 80%, at least 90%, preferably at least95%, more preferably at least 97%, or even more preferably at least 99%sequence identity to the sequence of the given antigen. By way ofexample, an Ab that binds specifically to human PD-1 may also havecross-reactivity with PD-1 antigens from certain primate species but maynot cross-react with PD-1 antigens from certain rodent species or withan antigen other than PD-1, e.g., a human PD-L1 antigen.

An immunoglobulin may derive from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. IgGsubclasses are also well known to those in the art and include but arenot limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to theAb class or subclass (e.g., IgM or IgG1) that is encoded by the heavychain constant region genes. The term “antibody” includes, by way ofexample, both naturally occurring and non-naturally occurring Abs;monoclonal and polyclonal Abs; chimeric and humanized Abs; human ornonhuman Abs; wholly synthetic Abs; and single chain Abs. A nonhuman Abmay be humanized by recombinant methods to reduce its immunogenicity inman. Where not expressly stated, and unless the context indicatesotherwise, the term “antibody” also includes an antigen-binding fragmentor an antigen-binding portion of any of the aforementionedimmunoglobulins, and includes a monovalent and a divalent fragment orportion, and a single chain Ab.

An “isolated antibody” refers to an Ab that is substantially free ofother Abs having different antigenic specificities (e.g., an isolated Abthat binds specifically to PD-1 is substantially free of Abs that bindspecifically to antigens other than PD-1). An isolated Ab that bindsspecifically to PD-1 may, however, have cross-reactivity to otherantigens, such as PD-1 molecules from different species. Moreover, anisolated Ab may be substantially free of other cellular material and/orchemicals. By comparison, an “isolated” nucleic acid refers to a nucleicacid composition of matter that is markedly different, i.e., has adistinctive chemical identity, nature and utility, from nucleic acids asthey exist in nature. For example, an isolated DNA, unlike native DNA,is a free-standing portion of a native DNA and not an integral part of alarger structural complex, the chromosome, found in nature. Further, anisolated DNA, unlike native genomic DNA, can typically be used inapplications or methods for which native genomic DNA is unsuited, e.g.,as a PCR primer or a hybridization probe for, among other things,measuring gene expression and detecting biomarker genes or mutations fordiagnosing disease or assessing the efficacy of a therapeutic. Anisolated nucleic acid may be purified so as to be substantially free ofother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, using standard techniques well known in theart. Examples of isolated nucleic acids include fragments of genomicDNA, PCR-amplified DNA, cDNA and RNA.

The term “monoclonal antibody” (“mAb”) refers to a preparation of Abmolecules of single molecular composition, i.e., Ab molecules whoseprimary sequences are essentially identical, and which exhibits a singlebinding specificity and affinity for a particular epitope. A mAb is anexample of an isolated Ab. MAbs may be produced by hybridoma,recombinant, transgenic or other techniques known to those skilled inthe art.

A “human” antibody (HuMAb) refers to an Ab having variable regions inwhich both the framework and CDR regions are derived from human germlineimmunoglobulin sequences. Furthermore, if the Ab contains a constantregion, the constant region also is derived from human germlineimmunoglobulin sequences. The human Abs of the invention may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody,” as used herein, is not intended to include Abs inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” Abs and “fully human” Abs and are usedsynonymously.

A “humanized” antibody refers to an Ab in which some, most or all of theamino acids outside the CDR domains of a non-human Ab are replaced withcorresponding amino acids derived from human immunoglobulins. In oneembodiment of a humanized form of an Ab, some, most or all of the aminoacids outside the CDR domains have been replaced with amino acids fromhuman immunoglobulins, whereas some, most or all amino acids within oneor more CDR regions are unchanged. Small additions, deletions,insertions, substitutions or modifications of amino acids arepermissible as long as they do not abrogate the ability of the Ab tobind to a particular antigen. A “humanized” Ab retains an antigenicspecificity similar to that of the original Ab.

A “chimeric antibody” refers to an Ab in which the variable regions arederived from one species and the constant regions are derived fromanother species, such as an Ab in which the variable regions are derivedfrom a mouse Ab and the constant regions are derived from a human Ab.

An “antigen-binding portion” of an Ab (also called an “antigen-bindingfragment”) refers to one or more fragments of an Ab that retain theability to bind specifically to the antigen bound by the whole Ab.

A “cancer” refers a broad group of various diseases characterized by theuncontrolled growth of abnormal cells in the body. Unregulated celldivision and growth divide and grow results in the formation ofmalignant tumors that invade neighboring tissues and may alsometastasize to distant parts of the body through the lymphatic system orbloodstream.

An “immune response” refers to the action of a cell of the immune system(for example, T lymphocytes, B lymphocytes, natural killer (NK) cells,macrophages, eosinophils, mast cells, dendritic cells and neutrophils)and soluble macromolecules produced by any of these cells or the liver(including Abs, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom a vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues.

An “immunoregulator” refers to a substance, an agent, a signalingpathway or a component thereof that regulates an immune response.“Regulating,” “modifying” or “modulating” an immune response refers toany alteration in a cell of the immune system or in the activity of suchcell. Such regulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunoregulators have been identified,some of which may have enhanced function in the cancer microenvironment.

The term “immunotherapy” refers to the treatment of a subject afflictedwith, or at risk of contracting or suffering a recurrence of, a diseaseby a method comprising inducing, enhancing, suppressing or otherwisemodifying an immune response. “Treatment” or “therapy” of a subjectrefers to any type of intervention or process performed on, or theadministration of an active agent to, the subject with the objective ofreversing, alleviating, ameliorating, inhibiting, slowing down orpreventing the onset, progression, development, severity or recurrenceof a symptom, complication, condition or biochemical indicia associatedwith a disease.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

A “predetermined threshold value,” relating to cell surface PD-L1expression, refers to the proportion of cells in a test tissue samplecomprising tumor cells and tumor-infiltrating inflammatory cells abovewhich the sample is scored as being positive for cell surface PD-L1expression. For cell surface expression assayed by IHC with the mAb28-8, the predetermined threshold value for cells expressing PD-L1 onthe cell surface ranges from at least about 0.01% to at least about 20%of the total number of cells. In preferred embodiments, thepredetermined threshold value for cells expressing PD-L1 on the cellsurface ranges from at least about 0.1% to at least about 10% of thetotal number of cells. More preferably, the predetermined thresholdvalue is at least about 5%. Even more preferably, the predeterminedthreshold value is at least about 1%, or in the range of 1-5%.

The “Programmed Death-1 (PD-1)” receptor refers to an immunoinhibitoryreceptor belonging to the CD28 family. PD-1 is expressed predominantlyon previously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. U64863.

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulateT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7.

A “signal transduction pathway” or “signaling pathway” refers to thebiochemical relationship between a variety of signal transductionmolecules that play a role in the transmission of a signal from oneportion of a cell to another portion of the cell. A “cell surfacereceptor” includes, for example, molecules and complexes of moleculesthat are located on the surface of a cell and are capable of receiving asignal and transmitting such a signal across the plasma membrane of acell. An example of a cell surface receptor of the present invention isthe PD-1 receptor, which is located on the surface of activated T cells,activated B cells and myeloid cells, and transmits a signal that resultsin a decrease in tumor-infiltrating lymphocytes and a decrease in T cellproliferation. An “inhibitor” of signaling refers to a compound or agentthat antagonizes or reduces the initiation, reception or transmission ofa signal, be that signal stimulatory or inhibitory, by any component ofa signaling pathway such as a receptor or its ligand.

A “subject” includes any human or nonhuman animal. The term “nonhumananimal” includes, but is not limited to, vertebrates such as nonhumanprimates, sheep, dogs, cats, rabbits and ferrets, rodents such as mice,rats and guinea pigs, avian species such as chickens, amphibians, andreptiles. In preferred embodiments, the subject is a mammal such as anonhuman primate, sheep, dog, cat, rabbit, ferret or rodent. In morepreferred embodiments, the subject is a human. The terms, “subject,”“patient” and “individual” are used interchangeably herein.

A “therapeutically effective amount” or “therapeutically effectivedosage” of a drug or therapeutic agent, such as an Ab of the invention,is any amount of the drug that, when used alone or in combination withanother therapeutic agent, protects a subject against the onset of adisease or promotes disease regression evidenced by a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction. The ability of a therapeuticagent to promote disease regression can be evaluated using a variety ofmethods known to the skilled practitioner, such as in human subjectsduring clinical trials, in animal model systems predictive of efficacyin humans, or by assaying the activity of the agent in in vitro assays.

By way of example, an anti-cancer agent promotes cancer regression in asubject. In preferred embodiments, a therapeutically effective amount ofthe drug promotes cancer regression to the point of eliminating thecancer. “Promoting cancer regression” means that administering aneffective amount of the drug, alone or in combination with ananti-neoplastic agent, results in a reduction in tumor growth or size,necrosis of the tumor, a decrease in severity of at least one diseasesymptom, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount of the drug preferably inhibits cell growth or tumorgrowth by at least about 20%, more preferably by at least about 40%,even more preferably by at least about 60%, and still more preferably byat least about 80% relative to untreated subjects. In other preferredembodiments of the invention, tumor regression may be observed andcontinue for a period of at least about 20 days, more preferably atleast about 40 days, or even more preferably at least about 60 days.Notwithstanding these ultimate measurements of therapeuticeffectiveness, evaluation of immunotherapeutic drugs must also makeallowance for “immune-related” response patterns.

An “immune-related” response pattern refers to a clinical responsepattern often observed in cancer patients treated with immunotherapeuticagents that produce antitumor effects by inducing cancer-specific immuneresponses or by modifying native immune processes. This response patternis characterized by a beneficial therapeutic effect that follows aninitial increase in tumor burden or the appearance of new lesions, whichin the evaluation of traditional chemotherapeutic agents would beclassified as disease progression and would be synonymous with drugfailure. Accordingly, proper evaluation of immunotherapeutic agents mayrequire long-term monitoring of the effects of these agents on thetarget disease.

A therapeutically effective amount of a drug includes a“prophylactically effective amount,” which is any amount of the drugthat, when administered alone or in combination with an anti-neoplasticagent to a subject at risk of developing a cancer (e.g., a subjecthaving a pre-malignant condition) or of suffering a recurrence ofcancer, inhibits the development or recurrence of the cancer. Inpreferred embodiments, the prophylactically effective amount preventsthe development or recurrence of the cancer entirely. “Inhibiting” thedevelopment or recurrence of a cancer means either lessening thelikelihood of the cancer's development or recurrence, or preventing thedevelopment or recurrence of the cancer entirely.

A “tumor-infiltrating inflammatory cell” is any type of cell thattypically participates in an inflammatory response in a subject andwhich infiltrates tumor tissue. Such cells include tumor-infiltratinglymphocytes (TILs), macrophages, monocytes, eosinophils, histiocytes anddendritic cells.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values or compositions areprovided in the application and claims, unless otherwise stated, themeaning of “about” or “comprising essentially of” should be assumed tobe within an acceptable error range for that particular value orcomposition.

As described herein, any concentration range, percentage range, ratiorange or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Various aspects of the invention are described in further detail in thefollowing subsections.

Antibodies of the Invention

Abs of the present invention include a variety of Abs having structuraland functional properties described herein, including high-affinitybinding to PD-1 or PD-L1, respectively. These Abs may be used, forexample, as therapeutic Abs to treat subjects afflicted with disease oras reagents in diagnostic assays to detect their cognate antigens. HumanmAbs (HuMAbs) that bind specifically to PD-1 (e.g., bind to human PD-1and may cross-react with PD-1 from other species, such as cynomolgusmonkey) with high affinity have been disclosed in U.S. Pat. No.8,008,449, and HuMAbs that bind specifically to PD-L1 with high affinityhave been disclosed in U.S. Pat. No. 7,943,743. The Abs of the inventioninclude, but are not limited to, all of the anti-PD-1 and anti-PD-L1 Absdisclosed in U.S. Pat. Nos. 8,008,449 and 7,943,743, respectively. Otheranti-PD-1 mAbs have been described in, for example, U.S. Pat. Nos.6,808,710, 7,488,802 and 8,168,757, and PCT Publication No. WO2012/145493, and anti-PD-L1 mAbs have been described in, for example,U.S. Pat. Nos. 7,635,757 and 8,217,149, U.S. Publication No.2009/0317368, and PCT Publication Nos. WO 2011/066389 and WO2012/145493. To the extent these anti-PD-1 and anti-PD-L1 mAbs exhibitthe structural and functional properties disclosed herein for antibodiesof the invention, they too are included as antibodies of the invention.

Anti-PD-1 Antibodies of the Invention

Each of the anti-PD-1 HuMAbs disclosed in U.S. Pat. No. 8,008,449 hasbeen demonstrated to exhibit one or more of the followingcharacteristics: (a) binds to human PD-1 with a K_(D) of 1×10⁻⁷ M orless, as determined by surface plasmon resonance using a Biacorebiosensor system; (b) does not substantially bind to human CD28, CTLA-4or ICOS; (c) increases T-cell proliferation in a Mixed LymphocyteReaction (MLR) assay; (d) increases interferon-γ production in an MLRassay; (e) increases IL-2 secretion in an MLR assay; (f) binds to humanPD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1and/or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses;(i) stimulates Ab responses; and (j) inhibits tumor cell growth in vivo.Anti-PD-1 Abs of the present invention include mAbs that bindspecifically to human PD-1 and exhibit at least one, preferably at leastfive, of the preceding characteristics.

U.S. Pat. No. 8,008,449 exemplifies seven anti-PD-1 HuMAbs: 17D8, 2D3,4H1, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3and 5F4. Isolated DNA molecules encoding the heavy and light chainvariable regions of these Abs have been sequenced, from which the aminoacid sequences of the variable regions were deduced. The V_(H) aminoacid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are providedherein as SEQ ID NOs. 1, 2, 3, 4, 5, 6 and 7, respectively. The V_(L)amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 areprovided herein as SEQ ID NOs. 8, 9, 10, 11, 12, 13 and 14,respectively.

Preferred anti-PD-1 Abs of the present invention include the anti-PD-1HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. These preferred Abs bindspecifically to human PD-1 and comprise: (a) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 1 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 8; (b) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 2 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 9; (c) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 3 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 10; (d) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 4 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 11; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 5 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 12; (f) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 6 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 13; or (g) a humanheavy chain variable region comprising consecutively linked amino acidshaving the sequence set forth in SEQ ID NO: 7 and a human light chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 14.

Given that each of these Abs can bind to PD-1, the V_(H) and V_(L)sequences can be “mixed and matched” to create other anti-PD-1 Abs ofthe invention. PD-1 binding of such “mixed and matched” Abs can betested using binding assays, e.g., enzyme-linked immunosorbent assays(ELISAs), western blots, radioimmunoassays and Biacore analysis that arewell known in the art (see, e.g., U.S. Pat. No. 8,008,449). Preferably,when V_(H) and V_(L) chains are mixed and matched, a V_(H) sequence froma particular V_(H)/V_(L) pairing is replaced with a structurally similarV_(H) sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence. Accordingly, anti-PD-1 Abs of the invention include anisolated mAb or antigen-binding portion thereof comprising: (a) a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs. 1, 2, 3, 4, 5, 6 and 7, and (b) alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs. 8, 9, 10, 11, 12, 13 and 14,wherein the Ab specifically binds PD-1, preferably human PD-1.

The CDR domains of the above Abs have been delineated using the Kabatsystem, and these Abs may also be defined by combinations of their 3heavy chain and 3 light chain CDRs (see U.S. Pat. No. 8,008,449). Sinceeach of these Abs can bind to PD-1 and antigen-binding specificity isprovided primarily by the CDR1, CDR2, and CDR3 regions, the V_(H) CDR1,CDR2, and CDR3 sequences and V_(κ) CDR1, CDR2, and CDR3 sequences can be“mixed and matched” (i.e., CDRs from different Abs can be mixed andmatch, although each Ab must contain a V_(H) CDR1, CDR2, and CDR3 and aV_(κ) CDR1, CDR2, and CDR3) to create other anti-PD-1 Abs that alsoconstitute Abs of the invention. PD-1 binding of such “mixed andmatched” Abs can be tested using the binding assays described above(e.g., ELISAs, western blots, radioimmunoassays and Biacore analysis).

Abs of the invention also include isolated Abs that bind specifically toPD-1 and comprise a heavy chain variable region derived from aparticular germline heavy chain immunoglobulin and/or a light chainvariable region derived from a particular germline light chainimmunoglobulin. Specifically, in certain embodiments, Abs of theinvention include isolated Abs comprising: (a) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-33 or 4-39 germline sequence, and/or alight chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L6, or L15 germlinesequence. The amino acid sequences of the V_(H) and V_(κ) regionsencoded by the V_(H) 3-33, V_(H) 4-39, V_(κ) L6 and V_(κ) L15 germlinegenes are provided in U.S. Pat. No. 8,008,449.

As used herein, an Ab can be identified as comprising a heavy or a lightchain variable region that is “derived from” a particular human germlineimmunoglobulin by comparing the amino acid sequence of the human Ab tothe amino acid sequences encoded by human germline immunoglobulin genes,and selecting the human germline immunoglobulin sequence that is closestin sequence (i.e., greatest percentage of sequence identity) to thesequence of the human Ab. A human Ab that is “derived from” a particularhuman germline immunoglobulin may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a selected human Ab is generally atleast 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human Ab as being human when compared to thegermline immunoglobulin amino acid sequences of other species (e.g.,murine germline sequences). In certain cases, a human Ab may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene.

In certain embodiments, the sequence of a human Ab derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In other embodiments, the human Ab maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifferences from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Preferred Abs of the invention also include isolated Abs orantigen-binding portions thereof comprising: (a) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-33 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L6 germline sequence; or (b) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 4-39 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L15 germlinesequence. Examples of Abs having a V_(H) and a V_(κ) derived from V_(H)3-33 and V_(κ) L6 germline sequences, respectively, include 17D8, 2D3,4H1, 5C4, and 7D3. Examples of Abs having V_(H) and V_(κ) regionsderived from V_(H) 4-39 and V_(κ) L15 germline sequences, respectively,include 4A11 and 5F4.

In yet other embodiments, anti-PD-1 Abs of the invention comprise heavyand light chain variable regions having amino acid sequences that arehighly similar or homologous to the amino acid sequences of thepreferred anti-PD-1 Abs described herein, wherein the Ab retains thefunctional properties of the preferred anti-PD-1 Abs of the invention.For example, Abs of the invention include mAbs comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises consecutively linked amino acids havinga sequence that is at least 80% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs. 1, 2, 3, 4, 5, 6 and7, and the light chain variable region comprises consecutively linkedamino acids having a sequence that is at least 80% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs. 8, 9,10, 11, 12, 13 and 14. In other embodiments, the V_(H) and/or V_(L)amino acid sequences may exhibit at least 85%, 90%, 95%, 96%, 97%, 98%or 99% identity to the sequences set forth above.

As used herein, the percent sequence identity (also referred to as thepercent sequence homology) between two sequences (amino acid ornucleotide sequences) is a function of the number of identical positionsshared by the sequences relative to the length of the sequences compared(i.e., % identity=number of identical positions/total number ofpositions being compared×100), taking into account the number of anygaps, and the length of each such gap, introduced to maximize the degreeof sequence identity between the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using mathematical algorithms that are well know tothose of ordinary skill in the art (see, e.g., U.S. Pat. No. 8,008,449).

Antibodies having very similar amino acid sequences are likely to haveessentially the same functional properties where the sequencedifferences are conservative modifications. As used herein,“conservative sequence modifications” refer to amino acid modificationsthat do not significantly affect the binding characteristics of the Abcontaining the amino acid sequence. Such conservative modificationsinclude amino acid substitutions, additions and deletions. Conservativeamino acid substitutions are substitutions in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Thus, for example, one or more amino acid residues within the CDRregions of an Ab of the invention can be replaced with other amino acidresidues from the same side chain family and the altered Ab can betested for retained function using functional assays that are well knownin the art. Accordingly, certain embodiments of the anti-PD-1 Abs of theinvention comprise heavy and light chain variable regions eachcomprising CDR1, CDR2 and CDR3 domains, wherein one or more of these CDRdomains comprise consecutively linked amino acids having sequences thatare the same as the CDR sequences of the preferred anti-PD-1 Absdescribed herein (e.g., 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4), orconservative modifications thereof, and wherein the Abs retain thedesired functional properties of the preferred anti-PD-1 Abs of theinvention.

Further, it is well known in the art that the heavy chain CDR3 is theprimary determinant of binding specificity and affinity of an Ab, andthat multiple Abs can predictably be generated having the same bindingcharacteristics based on a common CDR3 sequence (see, e.g., Klimka etal., 2000; Beiboer et al., 2000; Rader et al., 1998; Barbas et al.,1994; Barbas et al., 1995; Ditzel et al., 1996; Berezov et al., 2001;Igarashi et al., 1995; Bourgeois et al., 1998; Levi et al., 1993;Polymenis and Stoller, 1994; and Xu and Davis, 2000). The foregoingpublications demonstrate that, in general, once the heavy chain CDR3sequence of a given Ab is defined, variability in the other five CDRsequences does not greatly affect the binding specificity of that Ab.Thus, Abs of the invention comprising 6 CDRs can be defined byspecifying the sequence of the heavy chain CDR3 domain.

Anti-PD-1 Abs of the invention also include isolated Abs that bindspecifically to human PD-1 and cross-compete for binding to human PD-1with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. Thus,anti-PD-1 Abs of the invention include isolated Abs or antigen-bindingportions thereof that cross-compete for binding to PD-1 with a referenceAb or a reference antigen-binding portion thereof comprising: (a) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 1 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 8; (b) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 2 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 9; (c) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 3 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 10; (d) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 4 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 11; (e) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 5 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 12; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 6 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 13; or (g) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 7 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 14.

The ability of Abs to cross-compete for binding to an antigen indicatesthat these Abs bind to the same epitope region (i.e., the same or anoverlapping or adjacent epitope) of the antigen and sterically hinderthe binding of other cross-competing Abs to that particular epitoperegion. Thus, the ability of a test Ab to competitively inhibit thebinding of, for example, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, to humanPD-1 demonstrates that the test Ab binds to the same epitope region ofhuman PD-1 as 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, respectively. Allisolated Abs that bind to the same epitope region of human PD-1 as doesHuMAb 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 are included among the Absof the invention. These cross-competing Abs are expected to have verysimilar functional properties by virtue of their binding to the sameepitope region of PD-1. For example, cross-competing anti-PD-1 mAbs 5C4,2D3, 7D3, 4H1 and 17D8 have been shown to have similar functionalproperties (see U.S. Pat. No. 8,008,449 at Examples 3-7). The higher thedegree of cross-competition, the more similar will the functionalproperties be. Further, cross-competing Abs can be readily identifiedbased on their ability to cross-compete with 17D8, 2D3, 4H1, 5C4, 4A11,7D3 or 5F4 in standard PD-1 binding assays. For example, Biacoreanalysis, ELISA assays or flow cytometry may be used to demonstratecross-competition with the Abs of the invention (see, e.g., Examples 1and 2).

In certain embodiments, the Abs that cross-compete for binding to humanPD-1 with, or bind to the same epitope region of human PD-1 as, 17D8,2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 are mAbs. For administration to humanpatients, these cross-competing Abs are preferably chimeric Abs, or morepreferably humanized or human Abs. Such human mAbs can be prepared andisolated as described in U.S. Pat. No. 8,008,449. Data provided inExample 1 show that 5C4 or a Fab fragment thereof cross-competes witheach of 2D3, 7D3, 4H1 or 17D8 for binding to hPD-1 expressed on thesurface of a cell, indicating that all five anti-PD-1 mAbs bind to thesame epitope region of hPD-1 (FIGS. 1A-1C).

An anti-PD-1 Ab of the invention further can be prepared using an Abhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified Ab, which modified Ab mayhave altered properties from the starting Ab. An Ab can be engineered bymodifying one or more residues within one or both variable regions(i.e., V_(H) and/or V_(L)), for example within one or more CDR regionsand/or within one or more framework regions. Additionally oralternatively, an Ab can be engineered by modifying residues within theconstant region(s), for example, to alter the effector function(s) ofthe Ab. Specific modifications to Abs include CDR grafting,site-specific mutation of amino acid residues within the V_(H) and/orV_(κ) CDR1, CDR2 and/or CDR3 regions to thereby improve one or morebinding properties (e.g., affinity) of the Ab, site-specific mutation ofamino acid residues within the V_(H) and/or V_(κ) framework regions todecrease the immunogenicity of the Ab, modifications within the Fcregion, typically to alter one or more functional properties of the Ab,such as serum half-life, complement fixation, Fc receptor binding,and/or antigen-dependent cellular cytotoxicity, and chemicalmodification such as pegylation or alteration in glycosylation patternsto increase or decrease the biological (e.g., serum) half life of theAb. Specific examples of such modifications and methods of engineeringAbs are described in detail in U.S. Pat. No. 8,008,449. Anti-PD-1 Abs ofthe invention include all such engineered Abs that bind specifically tohuman PD-1 and are obtained by modification of any of theabove-described anti-PD-1 Abs.

Anti-PD-1 Abs of the invention also include antigen-binding portions ofthe above Abs. It has been amply demonstrated that the antigen-bindingfunction of an Ab can be performed by fragments of a full-length Ab.Examples of binding fragments encompassed within the term“antigen-binding portion” of an Ab include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; and (iv) a Fvfragment consisting of the V_(L) and V_(H) domains of a single arm of anAb.

These fragments, obtained initially through proteolysis with enzymessuch as papain and pepsin, have been subsequently engineered intomonovalent and multivalent antigen-binding fragments. For example,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker peptide that enables them to be made as a singleprotein chain in which the V_(L) and V_(H) regions pair to formmonovalent molecules known as single chain variable fragments (scFv).Divalent or bivalent scFvs (di-scFvs or bi-scFvs) can be engineered bylinking two scFvs in within a single peptide chain known as a tandemscFv which contains two V_(H) and two V_(L) regions. ScFv dimers andhigher multimers can also be created using linker peptides of fewer than10 amino acids that are too short for the two variable regions to foldtogether, which forces the scFvs to dimerize and produce diabodies orform other multimers. Diabodies have been shown to bind to their cognateantigen with much higher affinity than the corresponding scFvs, havingdissociation constants up to 40-fold lower than the K_(D) values for thescFvs. Very short linkers (≦3 amino acids) lead to the formation oftrivalent triabodies or tetravalent tetrabodies that exhibit even higheraffinities for to their antigens than diabodies. Other variants includeminibodies, which are scFv-C_(H3) dimers, and larger scFv-Fc fragments(scFv-C_(H2)-C_(H3) dimers), and even an isolated CDR may exhibitantigen-binding function. These Ab fragments are engineered usingconventional recombinant techniques known to those of skill in the art,and the fragments are screened for utility in the same manner as areintact Abs. All of the above proteolytic and engineered fragments of Absand related variants (see Hollinger and Hudson, 2005; Olafsen and Wu,2010, for further details) are intended to be encompassed within theterm “antigen-binding portion” of an Ab.

Anti-PD-L1 Antibodies of the Invention

Each of the anti-PD-L1 HuMAbs disclosed in U.S. Pat. No. 7,943,743 hasbeen demonstrated to exhibit one or more of the followingcharacteristics (a) binds to human PD-L1 with a K_(D) of 1×10⁻⁷ M orless; (b) increases T-cell proliferation in a Mixed Lymphocyte Reaction(MLR) assay; (c) increase interferon-γ production in an MLR assay; (d)increase IL-2 secretion in an MLR assay; (e) stimulates Ab responses;(f) inhibits the binding of PD-L1 to PD-1; and (g) reverses thesuppressive effect of T regulatory cells on T cell effector cells and/ordendritic cells. Anti-PD-L1 Abs of the present invention include mAbsthat bind specifically to human PD-L1 and exhibit at least one,preferably at least four, of the preceding characteristics.

U.S. Pat. No. 7,943,743 exemplifies ten anti-PD-1 HuMAbs: 3G10, 12A4(also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1,11E6, 12B7, and 13G4. Isolated DNA molecules encoding the heavy andlight chain variable regions of these Abs have been sequenced, fromwhich the amino acid sequences of the variable regions were deduced. TheV_(H) amino acid sequences of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1,11E6, 12B7, and 13G4 are shown in SEQ ID NOs. 15, 16, 17, 18, 19, 20,21, 22, 23 and 24, respectively, whereas their V_(L) amino acidsequences are shown in SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, 33and 34, respectively.

Preferred anti-PD-L1 Abs of the present invention include the anti-PD-L1HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4.These preferred Abs bind specifically to human PD-L1 and comprise: (a) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 15 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 25; (b) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 16 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 26; (c) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 17 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34.

Given that each of these Abs can bind to PD-L1, the V_(H) and V_(L)sequences can be “mixed and matched” to create other anti-PD-L1 Abs ofthe invention. PD-L1 binding of such “mixed and matched” Abs can betested using binding assays e.g., ELISAs, western blots,radioimmunoassays and Biacore analysis that are well known in the art(see, e.g., U.S. Pat. No. 7,943,743). Preferably, when V_(H) and V_(L)chains are mixed and matched, a V_(H) sequence from a particularV_(H)N_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, preferably a V_(L) sequence from a particularV_(H)N_(L) pairing is replaced with a structurally similar V_(L)sequence. Accordingly, Abs of the invention also include a mAb, orantigen binding portion thereof, comprising a heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in any of SEQ ID NOs. 15, 16, 17, 18, 19, 20, 21, 22, 23 or24, and a light chain variable region comprising consecutively linkedamino acids having the sequence set forth in any of SEQ ID NOs. 25, 26,27, 28, 29, 30, 31, 32, 33 or 34, wherein the Ab binds specifically toPD-L1, preferably human PD-L1.

The CDR domains of the above anti-PD-L1 HuMAbs have been delineatedusing the Kabat system, and these Abs may also be defined bycombinations of their 3 heavy chain and 3 light chain CDRs (see U.S.Pat. No. 7,943,743). Since each of these Abs can bind to PD-L1 andantigen-binding specificity is provided primarily by the CDR1, CDR2, andCDR3 regions, the V_(H) CDR1, CDR2, and CDR3 sequences and V_(κ) CDR1,CDR2, and CDR3 sequences can be “mixed and matched” (i.e., CDRs fromdifferent Abs can be mixed and match, although each Ab must contain aV_(H) CDR1, CDR2, and CDR3 and a V_(κ) CDR1, CDR2, and CDR3) to createother anti-PD-1 Abs that also constitute Abs of the invention. PD-L1binding of such “mixed and matched” Abs can be tested using, forexample, ELISAs, western blots, radioimmunoassays and Biacore analysis.

Antibodies of the invention also include Abs that bind specifically toPD-L1 and comprise a heavy chain variable region derived from aparticular germline heavy chain immunoglobulin and/or a light chainvariable region derived from a particular germline light chainimmunoglobulin. Specifically, in certain embodiments, Abs of theinvention include Abs comprising: (a) a heavy chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(H) 1-18, 1-69, 1-3 or 3-9 germline sequence, and/or alight chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L6, L15, A27 or L18germline sequence. The amino acid sequences of the V_(H) and V_(κ)regions encoded by the V_(H) 1-18, V_(H) 1-3, V_(H) 1-69, V_(H) 3-9,V_(κ) L6, V_(κ) L15 and V_(κ) A27 germline genes are provided in U.S.Pat. No. 7,943,743.

Preferred Abs of the invention include isolated Abs or antigen-bindingportions thereof comprising: (a) a heavy chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(H) 1-18 germline sequence, and a light chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(κ) L6 germline sequence; (b) a heavy chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(H) 1-69 germline sequence, and a lightchain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(κ) L6 germline sequence; (c) aheavy chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(H) 1-3 germlinesequence, and a light chain variable region that comprises consecutivelylinked amino acids having a sequence derived from a human V_(κ) L15germline sequence; (d) a heavy chain variable region that comprisesconsecutively linked amino acids having a sequence derived from a humanV_(H) 1-69 germline sequence, and a light chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(κ) A27 germline sequence; (e) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-9 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L15germline sequence; or (f) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 3-9 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(K) L18germline sequence.

An example of an Ab having a V_(H) and a V_(κ) derived from V_(H) 1-18and V_(κ) L6 germline sequences, respectively, is 3G10. Examples of Abshaving V_(H) and V_(κ) regions derived from V_(H) 1-69 and V_(κ) L6germline sequences, respectively, include 12A4, 1B12, 7H1 and 12B7. Anexample of an Ab having a V_(H) and a V_(κ) derived from V_(H) 1-3 andV_(κ) L15 germline sequences, respectively, is 10A5. Examples of Abshaving V_(H) and V_(κ) regions derived from V_(H) 1-69 and V_(κ) A27germline sequences, respectively, include 5F8, 11E6 and 11E6a. Anexample of an Ab having a V_(H) and a V_(κ) derived from V_(H) 3-9 andV_(κ) L15 germline sequences, respectively, is 10H10. An example of anAb having a V_(H) and a V_(κ) derived from V_(H) 1-3 and V_(κ) L15germline sequences, respectively, is 10A5. An example of an Ab having aV_(H) and a V_(κ) derived from V_(H) 3-9 and V_(κ) L18 germlinesequences, respectively, is 13G4.

In certain embodiments, anti-PD-L1 Abs of the invention comprise heavyand light chain variable regions having amino acid sequences that arehighly similar or homologous to the amino acid sequences of thepreferred anti-PD-L1 Abs described herein, wherein the Ab retains thefunctional properties of the aforementioned anti-PD-L1 Abs of theinvention. For example, Abs of the invention include mAbs comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises consecutively linked aminoacids having a sequence that is at least 80% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs. 15, 16, 17,18, 19, 20, 21, 22, 23, and 24, and the light chain variable regioncomprises consecutively linked amino acids having a sequence that is atleast 80% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. Inother embodiments, the V_(H) and/or V_(L) amino acid sequences mayexhibit at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to thesequences set forth above.

Certain embodiments of the anti-PD-L1 Abs of the invention compriseheavy and light chain variable regions each comprising CDR1, CDR2 andCDR3 domains, wherein one or more of these CDR domains compriseconsecutively linked amino acids having sequences that are the same asthe CDR sequences of the preferred anti-PD-L1 Abs described herein(e.g., 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4), orconservative modifications thereof, and wherein the Abs retain thedesired functional properties of the preferred anti-PD-L1 Abs of theinvention.

On the basis of the evidence that the heavy chain CDR3 is the primarydeterminant of binding specificity and affinity of an Ab, it isgenerally true that once the heavy chain CDR3 sequence of a given Ab isdefined, variability in the other five CDR sequences will not greatlyaffect the binding specificity of that Ab. Accordingly, anti-PD-L1 Absof the invention include isolated Abs comprising 6 CDRs, wherein the Absare defined by specifying the sequence of the heavy chain CDR3 domain.

Anti-PD-L1 Abs of the invention also include isolated Abs that bindspecifically to human PD-L1 and cross-compete for binding to human PD-L1with any of HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7and 13G4. Thus, anti-PD-L1 Abs of the invention include isolated Abs orantigen-binding portions thereof that cross-compete for binding to PD-L1with a reference Ab or a reference antigen-binding portion thereofcomprising: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 15 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 25; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 16 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 26; (c) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 17 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEO ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34.

The ability of an Ab to cross-compete with any of 3G10, 12A4, 10A5, 5F8,10H10, 1B12, 7H1, 11E6, 12B7 and 13G4 for binding to human PD-L1demonstrates that such Ab binds to the same epitope region of each of3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4,respectively. All isolated Abs that bind to the same epitope region ofhuman PD-L1 as does HuMAb 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6,12B7 or 13G4 are included among the Abs of the invention. Thesecross-competing Abs are expected to have very similar functionalproperties by virtue of their binding to the same epitope region ofPD-L1. For example, cross-competing anti-PD-L1 mAbs 3G10, 1B12, 13G4,12A4 (BMS-936559), 10A5, 12B7, 11E6 and 5F8 have been shown to havesimilar functional properties (see U.S. Pat. No. 7,943,743 at Examples3-11), whereas mAb 10H10, which binds to a different epitope region,behaves differently (U.S. Pat. No. 7,943,743 at Example 11). The higherthe degree of cross-competition, the more similar will the functionalproperties be. Further, cross-competing Abs can be identified instandard PD-L1 binding assays, e.g., Biacore analysis, ELISA assays orflow cytometry, that are well known to persons skilled in the art. Inpreferred embodiments, the Abs that cross-compete for binding to humanPD-1 with, or bind to the same epitope region of human PD-L1 as, 3G10,12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 are mAbs,preferably chimeric Abs, or more preferably humanized or human Abs. Suchhuman mAbs can be prepared and isolated as described in U.S. Pat. No.7,943,743.

Data provided in Example 2 show that each of the anti-PD-L1 HuMAbs 5F8,7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7 and 13G4, i.e., all of theHuMAbs tested except 10H10, substantially blocked binding of mAbs 3G10,10A5, 11E6, 12A4 and 13G4 to Chinese Hamster Ovary (CHO) cellsexpressing PD-L1 cells. HuMAb 10H10 substantially blocked the bindingonly of itself to CHO/PD-L1 cells. These data show that 3G10, 10A5,11E6, 12A4 and 13G4 cross-compete with all of the HuMAbs tested, exceptfor 10H10, for binding to the same epitope region of human PD-L1 (FIGS.2A-F).

Data provided in Example 3 show that the binding of HuMAb 12A4 to ES-2ovarian carcinoma cells expressing PD-L1 cells was substantially blockedby 12A4 itself and by 1B12 and 12B7, and was moderately to significantlyblocked by mAbs 5F8, 10A5, 13G4 and 3G10, but was not blocked by mAb10H10. These data, largely consistent with the data in Example 2, showthat 12A4 itself, and 2 other HuMabs, 12B7 and 1B12, substantiallycross-compete with 12A4 for binding to the same epitope region, possiblythe same epitope, of human PD-L1; 5F8, 10A5, 13G4 and 3G10, exhibit asignificant but lower level of cross-competition with 12A4, suggestingthat these mAbs may bind to epitopes that overlap the 12A4 epitope;whereas 10H10 does not cross-compete at all with 12A4 (FIG. 3),suggesting that this mAb binds to a different epitope region from 12A4.

Anti-PD-L1 Abs of the invention also include Abs engineered startingfrom Abs having one or more of the V_(H) and/or V_(L) sequencesdisclosed herein, which engineered Abs may have altered properties fromthe starting Abs. An anti-PD-L1 Ab can be engineered by a variety ofmodifications as described above for the engineering of modifiedanti-PD-1 Abs of the invention.

Anti-PD-L1 Abs of the invention also include isolated Abs selected fortheir ability to bind to PD-L1 in formalin-fixed, paraffin-embedded(FFPE) tissue specimens. The use of FFPE samples is essential for thelong-term follow-up analysis of the correlation between PD-L1 expressionin tumors and disease prognosis or progression. Yet, studies onmeasuring PD-L1 expression have often been conducted on frozen specimensbecause of the difficulty in isolating anti-human PD-L1 Abs that can beused to stain PD-L1 in FFPE specimens by IHC in general (Hamanishi etal., 2007) and, in particular, Abs that bind specifically to membranousPD-L1 in these tissues. The use of different Abs to stain PD-L1 infrozen versus FFPE tissues, and the ability of certain Abs todistinguish membranous and/or cytoplasmic forms of PD-L1, may accountfor some of the disparate data reported in the literature correlatingPD-L1 expression with disease prognosis (Hamanishi et al., 2007; Gadiotet al., 2011). This disclosure provides several rabbit mAbs that bindwith high affinity specifically to membranous human PD-L1 in FFPE tissuesamples comprising tumor cells and tumor-infiltrating inflammatorycells.

Rabbit and mouse anti-hPD-L1 mAbs were produced as described in Example9. Out of almost 200 Ab multiclones screened, only ten rabbit multicloneAbs were found to specifically detect the membranous form of PD-L1, andthe top five multiclones (designated Nos. 13, 20, 28, 29 and 49) weresubsequently subcloned. The clone that produced the most robustdetection specifically of membranous PD-L1, rabbit clone 28-8, wasselected for the IHC assays. The sequences of the variable regions ofmAb 28-8 are set forth in SEQ ID NOs. 35 and 36, respectively. Thesequences of the heavy and light chain CDR domains of mAb 28-8, asdelineated using the Kabat system, are set forth in SEQ ID NOs. 37-42.Rabbit clones 28-1, 28-12, 29-8 and 20-12 were the next best mAbs interms of robust detection of membranous PD-L1 in FFPE tissues.

Anti-PD-L1 Abs of the invention also include antigen-binding portions ofthe above Abs, including Fab, F(ab′)₂ Fd, Fv, and scFv, di-scFv orbi-scFv, and scFv-Fc fragments, diabodies, triabodies, tetrabodies, andisolated CDRs (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010, forfurther details).

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the present disclosure pertains to isolated nucleicacid molecules that encode any of the Abs of the invention. Thesenucleic acids may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. A nucleic acid of theinvention can be, for example, DNA or RNA, and may or may not containintronic sequences. In a preferred embodiment, the nucleic acid is acDNA.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For Abs expressed by hybridomas (e.g., hybridomasprepared from transgenic mice carrying human immunoglobulin genes asdescribed further below), cDNAs encoding the light and heavy chains ofthe Ab made by the hybridoma can be obtained by standard PCRamplification or cDNA cloning techniques. Nucleic acids encoding Absobtained from an immunoglobulin gene library (e.g., using phage displaytechniques) can be recovered from the library.

Preferred nucleic acids molecules of the invention are those encodingthe V_(H) and V_(κ) sequences of the anti-PD-1 HuMAbs, 17D8, 2D3, 4H1,5C4, 4A11, 7D3 and 5F4 (disclosed in U.S. Pat. No. 8,008,449), and thoseencoding the V_(H) and V_(κ) sequences of the anti-PD-L1 HuMAbs, 3G10,12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 (disclosed inU.S. Pat. No. 7,943,743). An isolated DNA encoding the V_(H) region canbe converted to a full-length heavy chain gene by operatively linkingthe V_(H)-encoding DNA to another DNA molecule encoding heavy chainconstant regions (C_(H1), C_(H2) and C_(H3)), the sequences of which areknown in the art and can be obtained by standard PCR amplification. Theheavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE,IgM or IgD constant region, but is preferably an IgG1 or IgG4 constantregion. Similarly, an isolated DNA encoding the V_(L) region can beconverted to a full-length light chain gene by operatively linking theV_(L)-encoding DNA to another DNA molecule encoding the light chainconstant region (C_(L)), the sequence of which is known in the art andcan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region, but most preferably isa kappa constant region.

Pharmaceutical Compositions

Antibodies of the present invention may be constituted in a composition,e.g., a pharmaceutical composition, containing one Ab or a combinationof Abs, or an antigen-binding portion(s) thereof, and a pharmaceuticallyacceptable carrier. As used herein, a “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Preferably,the carrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). A pharmaceutical composition of the invention may include oneor more pharmaceutically acceptable salts, anti-oxidant, aqueous andnonaqueous carriers, and/or adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents.

Dosage regimens are adjusted to provide the optimum desired response,e.g., a therapeutic response or minimal adverse effects. Foradministration of an anti-PD-1 or anti-PD-L1 Ab, the dosage ranges fromabout 0.0001 to about 100 mg/kg, usually from about 0.001 to about 20mg/kg, and more usually from about 0.01 to about 10 mg/kg, of thesubject's body weight. Preferably, the dosage is within the range of0.1-10 mg/kg body weight. For example, dosages can be 0.1, 0.3, 1, 3, 5or 10 mg/kg body weight, and more preferably, 0.3, 1, 3, or 10 mg/kgbody weight. The dosing schedule is typically designed to achieveexposures that result in sustained receptor occupancy (RO) based ontypical pharmacokinetic properties of an Ab. An exemplary treatmentregime entails administration once per week, once every two weeks, onceevery three weeks, once every four weeks, once a month, once every 3months or once every three to 6 months. The dosage and scheduling maychange during a course of treatment. For example, dosing schedule maycomprise administering the Ab: (i) every two weeks in 6-week cycles;(ii) every four weeks for six dosages, then every three months; (iii)every three weeks; (iv) 3-10 mg/kg body weight once followed by 1 mg/kgbody weight every 2-3 weeks. Considering that an IgG4 Ab typically has ahalf-life of 2-3 weeks, a preferred dosage regimen for an anti-PD-1 oranti-PD-L1 Ab of the invention comprises 0.3-10 mg/kg body weight,preferably 3-10 mg/kg body weight, more preferably 3 mg/kg body weightvia intravenous administration, with the Ab being given every 14 days inup to 6-week or 12-week cycles until complete response or confirmedprogressive disease.

In some methods, two or more mAbs with different binding specificitiesare administered simultaneously, in which case the dosage of each Abadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe, for example, weekly, every 2 weeks, every 3 weeks, monthly, everythree months or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of Ab to the target antigen in the patient. Insome methods, dosage is adjusted to achieve a plasma Ab concentration ofabout 1-1000 μg/ml and in some methods about 25-300 μg/ml.

Alternatively, the Ab can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the Ab in thepatient. In general, human Abs show the longest half-life, followed byhumanized Abs, chimeric Abs, and nonhuman Abs. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is typically administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being unduly toxic to the patient. Theselected dosage level will depend upon a variety of pharmacokineticfactors including the activity of the particular compositions of thepresent invention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A composition of the present invention can be administeredvia one or more routes of administration using one or more of a varietyof methods well known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

Uses and Methods of the Invention

The Abs, Ab compositions, nucleic acids and methods of the presentinvention have numerous in vitro and in vivo utilities including, forexample, methods to determine and quantify the expression of PD-1 orPD-L1 comprising binding of the Abs to the target polypeptides ormeasuring the amount of nucleic acid encoding these polypeptides, and amethod for immunotherapy of a subject afflicted with a diseasecomprising administering to the subject a composition comprising atherapeutically effective amount of a therapeutic agent that inhibitssignaling from an inhibitory immunoregulator. In preferred embodimentsof the latter method, the inhibitory immunoregulator is a component ofthe PD-1/PD-L1 signaling pathway, and the therapeutic agent disruptssignaling of this pathway. More preferably, the therapeutic agent is anAb that interferes with the interaction between PD-1 and PD-L1. Incertain preferred embodiments of this method, the Ab binds specificallyto PD-1 and blocks the interaction of PD-1 with PD-L1 and/or PD-L2. Inother preferred embodiments, the therapeutic agent is an Ab that bindsspecifically to PD-L1 and blocks the interaction of PD-L1 with PD-1and/or B7-1 (CD80). Thus, this disclosure provides methods forpotentiating an immune response in a subject comprising administering ananti-PD-1 and/or an anti-PD-L1 Ab in order to disrupt the interactionbetween PD-1 and PD-L1, and methods of treating diseases mediated bysuch a potentiation of the immune response. When Abs to PD-1 and PD-L1are administered together, the two can be administered sequentially ineither order or simultaneously. In certain aspects, this disclosureprovides methods of modifying an immune response in a subject comprisingadministering to the subject an anti-PD-1 and/or an anti-PD-L1 Ab of theinvention, or antigen-binding portion thereof, such that the immuneresponse in the subject is modified. Preferably, the immune response ispotentiated, enhanced, stimulated or up-regulated. In preferredembodiments, the Abs of the present invention are human Abs.

Preferred subjects include human patients in need of enhancement of animmune response. The immunotherapeutic methods disclosed herein areparticularly suitable for treating human patients having a disorder thatcan be treated by potentiating a T-cell mediated immune response. Incertain embodiments, the methods are employed for treatment of subjectsafflicted with a disease caused by an infectious agent. In preferredembodiments, the methods are employed for treatment of subjectsafflicted with, or at risk of being afflicted with, a cancer.

Cancer Immunotherapy

Blockade of PD-1/PD-L1 interaction has been shown to potentiate immuneresponses in vitro (U.S. Pat. Nos. 8,008,449 and 7,943,743; Fife et al.,2009) and mediate preclinical antitumor activity (Dong et al., 2002;Iwai et al., 2002). However, the molecular interactions potentiallyblocked by these two Abs are not identical: anti-PD-1 Abs of theinvention disrupt PD-1/PD-L1 and potentially PD-1/PD-L2 interactions; incontrast, whereas anti-PD-L1 Abs of the invention also disruptPD-1/PD-L1 interactions, they do not block PD-1/PD-L2 interactions butinstead may disrupt the PD-1-independent PD-L1/CD80 interaction, whichhas also been shown to down-modulate T-cell responses in vitro and invivo (Park et al., 2010; Paterson et al., 2011; Yang et al., 2011; Butteet al., 2007; Butte et al., 2008). Thus, it is possible that among thesevaried ligand-receptor pairings, different interactions may dominate indifferent cancer types, contributing to dissimilar activity profiles forthe two Abs.

Disruption of the PD-1/PD-L1 interaction by antagonistic Abs can enhancethe immune response to cancerous cells in a patient. PD-L1 is notexpressed in normal human cells, but is abundant in a variety of humancancers (Dong et al., 2002). The interaction between PD-1 and PD-L1impairs T cell responses as manifested by a decrease intumor-infiltrating lymphocytes (TILs) and a decrease in T-cellreceptor-mediated proliferation, resulting in T cell anergy, exhaustionor apoptosis, and immune evasion by the cancerous cells (Zou and Chen,2008; Blank et al., 2005; Konishi et al., 2004; Dong et al., 2003; Iwaiet al., 2002) Immune suppression can be reversed by inhibiting the localinteraction between PD-L1 and PD-1 using an anti-PD-1 and/or ananti-PD-L1 Ab. These Abs may be used alone or in combination to inhibitthe growth of cancerous tumors. In addition, either or both of these Absmay be used in conjunction with other immunogenic and/or anti-canceragents including cytokines, standard cancer chemotherapies, vaccines,radiation, surgery, or other Abs.

Immunotherapy of Cancer Patients Using an Anti-PD-1 Antibody

This disclosure provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises administering to thesubject a composition comprising a therapeutically effective amount ofan Ab or an antigen-binding portion thereof that disrupts theinteraction of PD-1 with PD-L1 and/or PD-L2. This disclosure alsoprovides a method of inhibiting growth of tumor cells in a subject,comprising administering to the subject an Ab or an antigen-bindingportion thereof that disrupts the interaction of PD-1 with PD-L1 and/orPD-L2 in an amount effective to inhibit growth of the tumor cells. Inpreferred embodiments, the subject is a human. In other preferredembodiments, the Ab or antigen-binding portion thereof is an anti-PD-1Ab of the invention or an antigen-binding portion thereof. In certainembodiments, the Ab or antigen-binding portion thereof is of an IgG1 orIgG4 isotype. In other embodiments, the Ab or antigen-binding portionthereof is a mAb or an antigen-binding portion thereof. In furtherembodiments, the Ab or antigen-binding portion thereof is a chimeric,humanized or human Ab or an antigen-binding portion thereof. Inpreferred embodiments for treating a human patient, the Ab orantigen-binding portion thereof is a human Ab or an antigen-bindingportion thereof.

The clinical trials described in the Examples employed the anti-PD-1HuMAb, nivolumab (designated 5C4 in U.S. Pat. No. 8,008,449), to treatcancer. While 5C4 was selected as the lead Ab for entering the clinic,it is notable that several anti-PD-1 Abs of the invention share with 5C4functional properties that are important to the therapeutic activity of5C4, including high affinity binding specifically to human PD-1,increasing T-cell proliferation, IL-2 secretion and interferon-γproduction in an MLR assay, inhibiting the binding of PD-L1 and/or PD-L2to PD-1, and inhibiting tumor cell growth in vivo. Moreover, certain ofthe anti-PD-1 Abs of the invention, 17D8, 2D3, 4H1 and 7D3 arestructurally related to 5C4 in comprising V_(H) and V_(κ) regions thathave sequences derived from V_(H) 3-33 and V_(κ) L6 germline sequences,respectively. In addition, 5C4, 2D3, 7D3, 4H1 and 17D8 all cross-competefor binding to the same epitope region of hPD-1 (Example 1). Thus, thepreclinical characterization of nivolumab and other anti-PD-1 HuMabsindicate that the methods of treating cancer provided herein may beperformed using different Abs selected from the broad genus of anti-PD-1Abs of the invention.

Accordingly, certain embodiments of the immunotherapy methods disclosedherein comprise administering to a patient an anti-PD-1 Ab orantigen-binding portion thereof comprising: (a) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 3-33 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L6 germline sequence, or (b) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 4-39 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L15 germlinesequence.

In certain other embodiments, the Ab or antigen-binding portion thereofthat is administered to the patient cross-competes for binding to PD-1with a reference Ab or a reference antigen-binding portion thereofcomprising: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 1 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 8; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 2 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 9; (c) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 3 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 10; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 4 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 11; (e) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 5 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 12; (f) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 6 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 13; or (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 7 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 14. In preferred embodiments, the Ab or antigen-binding portionthereof cross-competes for binding to PD-1 with nivolumab.

In certain embodiments of the immunotherapy methods disclosed herein,the anti-PD-1 Ab or antigen-binding portion thereof administered to thepatient comprises: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 1 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 8; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 2 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 9; (c) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 3 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 10; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 4 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 11; (e) a human heavy chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 5 and ahuman light chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 12; (f) a human heavychain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 6 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 13; or (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 7 and a human light chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 14. In preferred embodiments, the anti-PD-1 Ab or antigen-bindingportion comprises a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 4 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 11. Inother preferred embodiments, the anti-PD-1 Ab is nivolumab.

In certain embodiments of the present methods, the anti-PD-1 Ab isformulated for intravenous administration. In preferred embodiments, theAb is administered intravenously at a dose of 3 mg/kg over 60 minutesevery 2 weeks. Typically, treatment is continued as long as clinicalbenefit is observed or until unmanageable toxicity or diseaseprogression occurs.

In the clinical trials of anti-PD-1 immunotherapy described in theExamples below, intriguing ORs with durable clinical responses, even inheavily pretreated patients, were observed across multiple tumor typesincluding a substantial proportion of NSCLC, MEL, and RCC patients andin various sites of metastasis including liver, lung, lymph nodes, andbone. MEL and RCC are considered to be immunogenic neoplasms, havingpreviously been demonstrated to be responsive to cancer immunotherapy,e.g., interferon-alfa and interleukin-2 in both MEL and RCC (Eton etal., 2002; Coppin et al., 2005; McDermott and Atkins, 2006) andanti-CTLA-4 Ab in MEL (Hodi et al., 2010). In MEL patients, significantORRs were observed with nivolumab across different doses of 0.1, 0.3, 1,3, or 10 mg/kg with an overall response rate of about 31%, and similarlycomparable ORRs of about 30% were observed in RCC patients treated withnivolumab doses of 1 or 10 mg/kg (see Example 7). In contrast,immunotherapy has historically had minimal success in lung cancer. Thislack of success has been attributed to NSCLC being “non-immunogenic”(see, e.g., Holt and Disis, 2008; Holt et al., 2011). The ability oflung cancer to thwart the immune system results from multiple factors,including the secretion of immunosuppressive cytokines, loss of majorhistocompatibility complex antigen expression, and co-opting of T cellinhibitory pathways (Dasanu et al., 2012; Marincola et al., 2000;Brahmer et al, 2012).

Despite the limited efficacy of immunotherapy historically seen in lungcancer and the diverse mechanisms employed by lung cancer cells to evadeimmune system attack, various tumor cell vaccines (e.g.,belagenpumatucel-L, a whole-cell-based vaccine that blocks the action ofTGF-β2) and antigen-based vaccines (e.g., a humoral EGF vaccine andvaccines incorporating a MAGE-A3 fusion protein or portions of thetumor-associated MUC1 antigen) that augment antigen-specific antitumorimmunity are being evaluated in clinical trials (Holt et al., 2011;Shepherd et al., 2011; Dasanu et al., 2012; Brahmer et al, 2012).However, while such antigen-specific vaccines have shown some promisefor the treatment of NSCLC, Holt et al. (2011) noted that trialsinvolving nonspecific immunotherapeutic interventions had failed toimprove outcomes in NSCLC, which indicated a need to combine them withantigen-specific vaccines. These authors expressed the view that trulyefficacious immunotherapy of NSCLC would only result from implementationof strategies to both augment antitumor immunity and counteracttumor-mediated immunosuppression, and concluded that the nonexistent orweak nascent immune responses in NSCLC patients make it unlikely thatnonspecific stimulation of the immune system or removal ofimmunosuppression would create changes in clinical outcomes.

Thus, the results reported herein with NSCLC are particularly striking,unexpected and surprising. In NSCLC patients, ORs were observed atnivolumab doses of 1, 3, or 10 mg/kg with response rates of 3%, 24%, and20%, respectively (see Example 7). ORs were observed across NSCLChistologies: 9 responders of 54 squamous (17%), and 13 of 74 nonsquamous(18%). This level of activity seen with nivolumab in NSCLC patients withsignificant prior therapy (54% with 3 lines of previous therapy) andacross histologies is unprecedented, especially in the squamoushistology patients (cf. Gridelli et al., 2008; Miller, 2006), andprovides a very favorable benefit/risk dynamic regarding efficacy andsafety compared to existing standard-of-care.

In certain embodiments of the present immunotherapy methods, theanti-PD-1 Ab is indicated as monotherapy for locally advanced ormetastatic squamous or non-squamous NSCLC after platinum based therapy(regardless of maintenance) and one other treatment. In otherembodiments, the anti-PD-1 Ab is indicated as monotherapy for locallyadvanced or metastatic squamous or non-squamous NSCLC after failure ofplatinum-based therapy and one other prior chemotherapy regimen. Infurther embodiments, the anti-PD-1 Ab is indicated as monotherapy forlocally advanced or metastatic squamous or non-squamous NSCLC afterfailure of two lines of therapy one of which must include platinum basedregimen. In yet further embodiments, the anti-PD-1 Ab is indicated asmonotherapy for locally advanced or metastatic squamous or non-squamousNSCLC after at least one prior chemotherapy, after failure of at leastone prior platinum based therapy, or after progression on at least oneplatinum doublet therapy. In all of the immunotherapy methods disclosedherein, treatment may be continued as long as clinical benefit isobserved or until unmanageable toxicity or disease progression occurs.

Durability of Clinical Responses to Anti-PD-1 in Heavily-PretreatedCancer Patients

The critical role of the PD-1 pathway in suppressing anti-tumorimmunity, first revealed in laboratory models, has now been validated inclinical studies. As described herein, monotherapy with drugs blockingPD-1 (nivolumab) or its major ligand PD-L1 (BMS-936559) can mediateregression in patients with treatment-refractory advanced cancers. Theobjective response rate (ORR) in heavily pretreated NSCLC patientsreceiving anti-PD-1 Ab, including patients with squamous histology, issurprising and unexpected, as standard salvage therapies historicallyshow modest benefit in these patients (Scagliotti et al., 2011). Asmeasured by standard RECIST in this study, ORs were long-lasting, withmedian response durations of about 18 months in 22 of 129 responders((see Example 7). In addition, patterns of tumor regression consistentwith immune-related patterns of response were observed.

These findings have established the PD-1 pathway as a new therapeuticfocus in oncology (Pardoll, 2012; Topalian et al., 2012c; Hamid andCarvajal, 2013). In the current study, in which 54% of patients hadprogressive disease following 3 or more prior systemic regimens,preliminary analysis has been conducted up to March 2013. This updatedanalysis has supported and reinforced the data obtained and conclusionsreached from the earlier February 2012 analyses. Thus, conventional ORsor prolonged disease stabilization were documented in patients withNSCLC (17% and 10%, respectively), MEL (31%, 7%) and RCC (29%, 27%),across all doses tested (see Example 7). Additionally, 13 patients (4%)manifested unconventional, “immune-related” response patterns aspreviously described with anti-CTLA-4 therapy, several of which weresustained (Sharma et al., 2011).

The durability of ORs across multiple cancer types in patients treatedwith the anti-PD-1 Ab is particularly notable. The updated analysesagain underscored the durability of clinical activity innivolumab-treated patients, which has not generally been observed withchemotherapy or small molecule inhibitors to date, but has been observedin some patients with advanced melanoma receiving immunotherapies,including ipilimumab and high-dose interleukin-2 (Topalian et al., 2011;Hodi et al., 2010). The persistence of partial tumor regressionsfollowing drug discontinuation suggests that PD-1 blockade has reset theimmune equilibrium between tumor and host, and the OS benefit mayultimately prove to be significantly longer than has been measured todate. Further follow-up is needed to determine the ultimate durabilityof tumor regression and disease stabilization in patients on theseclinical trials.

Significantly, the durable objective tumor regression and diseasestabilization induced by nivolumab in heavily-pretreated patients withadvanced NSCLC, MEL, and RCC translate to survival outcomes that surpasshistorical data for these patient populations treated with conventionalchemotherapy and/or tyrosine-kinase inhibitor (TKI) treatments. InNSCLC, 1- and 2-year survival rates were 42% and 14%, respectively, withmedian OS of 9.2 and 10.1 months in patients with squamous andnon-squamous cancers, respectively (see Example 7). This high level ofefficacy is especially impressive since 54% of these patients hadreceived 3 or more prior therapies. Moreover, because follow-up of manylung cancer patients was relatively limited, these figures may change asdata mature. Historically, 2L chemotherapeutics for lung cancer (i.e.,docetaxel and pemetrexed) have achieved a median OS of 7.5-8.3 months,and one-year survival rates of approximately 30% (Shepherd et al., 2000;Hanna et al., 2004). In a 2L/3L population, erlotinib-treated patientshad a median survival of 6.7 months, versus 4.7 months inplacebo-treated patients (Shepherd et al., 2005). No therapy iscurrently approved for use in lung cancer beyond the 3L setting, andminimal data exist to benchmark survival in this patient population,except in retrospective reviews with reported median survivals of5.8-6.5 months and 1-year survivals of 25% (Girard et al., 2009;Scartozi et al., 2010).

In nivolumab-treated MEL patients, median OS of 16.8 months wasachieved, with landmark survival rates of 62% (1-year) and 43% (2-year)(see Example 7). Survival outcomes in pretreated melanoma patientssupported the recent FDA approvals of ipilimumab and vemurafenib. In arecent phase 3 trial enrolling melanoma patients with at least one priortreatment for metastatic disease, ipilimumab increased median OS from6.4 to 10.1 months, compared to a gp100 peptide vaccine (Hodi et al.,2010). In phase 2 trials of ipilimumab in previously treated patients,2-year survival rates ranged from 24.2-32.8% (Lebbe et al., 2012).Median OS in previously treated patients with BRAF-mutant melanomaenrolled on a large phase 2 of vemurafenib was 15.9 months, and 1-yearsurvival was 58% (Sosman et al., 2012).

Accordingly, in certain embodiments, immunotherapy with anti-PD-1 Ab isindicated as monotherapy for locally advanced or metastatic MEL aftertherapy with dacarbazine (regardless of maintenance) and one othertreatment. In other embodiments, the anti-PD-1 Ab is indicated asmonotherapy for locally advanced or metastatic MEL after failure ofdacarbazine-based therapy. In further embodiments, the anti-PD-1 Ab isindicated as monotherapy for locally advanced or metastatic MEL afterfailure of two lines of therapy one of which must include adacarbazine-based regimen. In yet further embodiments, the anti-PD-1 Abis indicated as monotherapy for locally advanced or metastatic MEL afterat least one prior chemotherapy, after failure of at least one priordacarbazine-based therapy, or after progression on at least dacarbazinetherapy. In all of the immunotherapy methods disclosed herein, treatmentmay be continued as long as clinical benefit is observed or untilunmanageable toxicity or disease progression occurs.

In nivolumab-treated patients with RCC, among whom 45% received 3 ormore prior therapies and 71% received prior anti-angiogenic therapy,median OS was reached at 22 months (as of the March 2013 date ofanalysis). Landmark survival rates of 70% (1-year) and 50% (2-year) wereobserved (see Example 7). In a recent Phase 3 trial enrolling kidneycancer patients whose disease progressed following anti-angiogenictherapy, everolimus was compared with placebo: median OS was 14.8 versus14.4 months, respectively (Motzer et al., 2008; Motzer et al., 2010). Arecent Phase 3 trial comparing sorafenib to temsirolimus in asunitinib-refractory kidney cancer population yielded median OS of 16.6and 12.3 months, respectively (Hutson et al., 2012). Thus, as with NSCLCand MEL, treatment of a heavily-pretreated RCC patient population withnivolumab has yielded a considerably longer median OS (>22 months) thantreatment of a less refractory population with standard-of-caretherapies. Controlled Phase 3 trials with prospective survival endpointsare underway in NSCLC, MEL and RCC (NCT01673867, NCT01721772,NCT01642004, NCT01668784, and NCT01721746 (see Clinical Trials Website,http://www.clinicaltrials.gov). The results from these trials areexpected to further demonstrate the high efficacy of, and durability ofresponses to, nivolumab in these cancers compared standard-of-caretherapies.

In certain embodiments, immunotherapy with anti-PD-1 Ab is indicated asmonotherapy for locally advanced or metastatic RCC after therapy with ananti-angiogenic TKI or a mTOR inhibitor (regardless of maintenance) andone other treatment. In other embodiments, the anti-PD-1 Ab is indicatedas monotherapy for locally advanced or metastatic RCC after failure oftherapy with an anti-angiogenic TKI or a mTOR inhibitor. In furtherembodiments, the anti-PD-1 Ab is indicated as monotherapy for locallyadvanced or metastatic RCC after failure of two lines of therapy one ofwhich must include an anti-angiogenic TKI or a mTOR inhibitor. In yetfurther embodiments, the anti-PD-1 Ab is indicated as monotherapy forlocally advanced or metastatic RCC after at least one priorchemotherapy, after failure of at least one prior anti-angiogenic TKI-or mTOR inhibitor-based therapy, or after progression on at least ananti-angiogenic TKI or a mTOR inhibitor therapy. Anti-PD-1 immunotherapymay be continued as long as clinical benefit is observed or untilunmanageable toxicity or disease progression occurs.

Notably, OS in lung cancer, melanoma and kidney cancer patientsreceiving nivolumab was considerably longer than PFS. These resultsmirror those reported for ipilimumab (Hodi et al., 2010), and reflectthe observation that early tumor enlargement or the appearance of newlesions in some patients receiving immune checkpoint blockade can evolveto disease stabilization or regression. These findings suggest thatprogression-free survival may not be an optimal endpoint for determiningthe efficacy of nivolumab and other agents in this class.

The data disclosed herein demonstrating the high efficacy, durabilityand broad applicability of anti-PD-1 immunotherapy for treating cancerhas led to nivolumab being tested for additional types of cancer. Forexample, on the basis that increased PD-L1 expression has been reportedwith various hematologic malignancies and may prevent the host immuneresponse from exerting a beneficial impact on the malignant cells, atrial to confirm the ability of nivolumab to mediate antitumor activityin patients with hematologic malignancies (multiple myeloma, B-celllymphoma, T-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-celllymphoma, and chronic myelogenous leukemia) has been initiated(NCT01592370). Nivolumab is also being tested as a monotherapy inadvanced hepatocellular carcinoma (NCT01658878).

In summary, the results of anti-PD-1 immunotherapy disclosed herein areremarkable in at least the following three respects. First, the efficacyof anti-PD-1 has been shown to surpass historical efficacy data forpatients on standard-of-care treatments for cancer. Notably, thisefficacy has been demonstrated in patient in heavily pretreatedpopulations in which about half of the patients had progressive diseasefollowing 3 or more prior systemic regimens. Such patients, afflictedwith advanced, metastatic and/or refractory cancers, are notoriouslydifficult to treat. Accordingly, this disclosure provides methods forimmunotherapy of a patient afflicted with an advanced, metastatic and/orrefractory cancer, which method comprises administering to the patient atherapeutically effective amount of an Ab or an antigen-binding portionthereof that disrupts the interaction of PD-1 with PD-L1 and/or PD-L2.In certain embodiments of any of the therapeutic methods disclosedherein, the subject has been pre-treated for the cancer; for example,the subject had undergone at least one, two, or three prior lines oftherapy for cancer.

Second, the present therapeutic methods have been shown to be applicableto a broad genus of different cancers. Based on the surprising discoverythat even a “non-immunogenic” cancer such as NSCLC (Holt et al., 2011)and hard-to-treat cancers such as ovarian and gastric cancers (as wellas other cancers tested, including MEL, RCC, and CRC) are amendable totreatment with anti-PD-1 and/or anti-PD-L1 (see Examples 7 and 14), thisdisclosure generally provides methods for immunotherapy of a patientafflicted with practically any of a very wide range of cancers.

Third, treatment with an anti-PD-1 or anti-PD-L1 Ab has been shown toproduce strikingly durable clinical activity in cancer patients.Accordingly, this disclosure provides immunotherapeutic methods ofinducing a durable clinical response in a cancer patient comprisingadministering to the patient a therapeutically effective amount of an Abor an antigen-binding portion thereof that disrupts the interaction ofPD-1 with PD-L1 and/or PD-L2. In preferred embodiments of any of thetherapeutic methods described herein, the clinical response is a durableresponse.

As used herein, a “durable” response is a therapeutic or clinicalresponse that exceeds the anticipated median OS rate in a patientpopulation. The anticipated median OS rate varies with different cancersand different patient populations. In certain embodiments, a durableresponse exceeds the anticipated median OS rate in the relevant patientpopulation by at least 10%, preferably by at least 20%, more preferablyby at least 30%, and even more preferably by at least 50%. A majorbenefit of immunotherapeutic approaches based on PD-1 pathway blockademay be the functional restoration of exhausted T cells with long-termgeneration of memory T cells that may maintain antitumor immunesurveillance and inhibit tumor growth for prolonged periods extending tomany years, even in the absence of continued therapy (Kim and Ahmed,2010). Indeed, long-term follow-up studies on patients followingcessation of nivolumab therapy have confirmed that a patient with CRCexperienced a complete response which was ongoing after 3 years; apatient with RCC experienced a partial response lasting 3 years offtherapy, which converted to a complete response that was ongoing at 12months; and a patient with melanoma achieved a partial response that wasstable for 16 months off therapy, and recurrent disease was successfullytreated with reinduction anti-PD-1 therapy (Lipson et al., 2013).

Immune-Related Clinical Responses

It has become evident that conventional response criteria may notadequately assess the activity of immunotherapeutic agents becauseprogressive disease (by initial radiographic evaluation) does notnecessarily reflect therapeutic failure. For example, treatment with theanti-CTLA-4 Ab, ipilimumab, has been shown to produce four distinctresponse patterns, all of which were associated with favorable survival:(a) shrinkage in baseline lesions, without new lesions; (b) durablestable disease (in some patients followed by a slow, steady decline intotal tumor burden); (c) response after an increase in total tumorburden; and (d) response in the presence of new lesions. Accordingly, toproperly evaluate immunotherapeutic agents, long-term effects on thetarget disease must also be captured. In this regard, systematicimmune-related response criteria (irRC) that make allowances for anearly increase in tumor burden and/or the appearance of new lesions, andwhich seek to enhance the characterization of immune-related responsepatterns, have been proposed (Wolchok et al., 2009). While the fullimpact of these unconventional response patterns remains to be definedin randomized trials of nivolumab with survival endpoints, the presentobservations are reminiscent of findings with ipilimumab in which asignificant extension of OS was observed in treated patients (Hodi etal., 2010; Robert et al., 2011).

The overall risk/benefit profile of anti-PD-1 immunotherapy is alsofavorable, with a low incidence of more severe drug-related adverseevents (AEs; >grade 3), the specific events observed to date beingconsistent with other immunotherapeutic agents. This suggests thatanti-PD-1 immunotherapy can be delivered in an outpatient setting withminimal supportive care.

Broad Spectrum of Cancers Treatable by Anti-PD-1 Immunotherapy

The clinical data presented herein demonstrate that immunotherapy basedon PD-1 blockade is not limited to only “immunogenic” tumor types, suchas MEL and RCC, but extends to tumor types not generally considered tobe immune-responsive, including NSCLC. The unexpected successes withtreatment-refractory metastatic NSCLC underscore the possibility thatany neoplasm can be “immunogenic” in the context of proper immunemodulation, and suggest that PD-1 blockade as an immunotherapeuticapproach is broadly applicable across a very diverse range of tumortypes. Thus, cancers that may be treated using the anti-PD-1 Abs of theinvention also include cancers typically responsive to immunotherapy aswell as cancers that have traditionally been regarded asnon-immunogenic. Non-limiting examples of preferred cancers fortreatment include NSCLC, MEL, RCC, CRC, CRPC, HCC, squamous cellcarcinoma of the head and neck, carcinomas of the esophagus, ovary,gastrointestinal tract and breast, and a hematologic malignancy.Although NSCLC is not generally considered responsive to immunotherapy,data disclosed herein unexpectedly demonstrate that both squamous andnon-squamous NSCLC are responsive to treatment with an anti-PD-1 Ab.Additionally, the disclosure provides for the treatment of refractory orrecurrent malignancies whose growth may be inhibited using an anti-PD-1Ab of the invention.

Examples of cancers that may be treated using an anti-PD-1 Ab in themethods of the present invention, based on the indications of very broadapplicability of anti-PD-1 immunotherapy provided herein, include livercancer, bone cancer, pancreatic cancer, skin cancer, cancer of the heador neck, breast cancer, lung cancer, cutaneous or intraocular malignantmelanoma, renal cancer, uterine cancer, ovarian cancer, colorectalcancer, colon cancer, rectal cancer, cancer of the anal region, stomachcancer, testicular cancer, uterine cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer ofthe esophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, solid tumors of childhood, lymphocyticlymphoma, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, environmentally induced cancers includingthose induced by asbestos, hematologic malignancies including, forexample, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primarymediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloidlymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia,follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides,anaplastic large cell lymphoma, T-cell lymphoma, and precursorT-lymphoblastic lymphoma, and any combinations of said cancers. Thepresent invention is also applicable to treatment of metastatic cancers.

Medical Uses of Anti-PD-1 Abs

One aspect of this invention is the use of any anti-PD-1 Ab orantigen-binding portion thereof of the invention for the preparation ofa medicament for inhibiting signaling from the PD-1/PD-L1 pathway so asto thereby potentiate an endogenous immune response in a subjectafflicted with cancer. Another aspect is the use of any anti-PD-1 Ab oran antigen-binding portion thereof of the invention for the preparationof a medicament for immunotherapy of a subject afflicted with cancercomprising disrupting the interaction between PD-1 and PD-L1. These usesfor the preparation of medicaments are broadly applicable to the fullrange of cancers disclosed herein. In preferred embodiments of theseuses, the cancers include squamous NSCLC, non-squamous NSCLC, MEL, RCC,CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, andcarcinomas of the esophagus, ovary, gastrointestinal tract and breast,and a hematologic malignancy. This disclosure also provides medical usesof any anti-PD-1 Ab or antigen-binding portion thereof of the inventioncorresponding to all the embodiments of the methods of treatmentemploying an anti-PD-1 Ab described herein.

The disclosure also provides an anti-PD-1 Ab or an antigen-bindingportion thereof of the invention for use in treating a subject afflictedwith cancer comprising potentiating an endogenous immune response in thesubject by inhibiting signaling from the PD-1/PD-L1 pathway. Thedisclosure further provides an anti-PD-1 Ab or an antigen-bindingportion thereof of the invention for use in immunotherapy of a subjectafflicted with cancer comprising disrupting the interaction between PD-1and PD-L1. These Abs may be used in potentiating an endogenous immuneresponse against, or in immunotherapy of, the full range of cancersdisclosed herein. In preferred embodiments, the cancers include squamousNSCLC, non-squamous NSCLC, MEL (e.g., metastatic malignant MEL), RCC,CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, andcarcinomas of the esophagus, ovary, gastrointestinal tract and breast,and a hematologic malignancy.

Combination Therapy Including Anti-PD-1 Antibodies

Whereas monotherapy with anti-PD-1 and anti-PD-L1 Abs has been shownherein to significantly increase the survival of patients with lungcancer, melanoma, kidney cancer, and potentially other malignancies,preclinical data indicate that synergistic treatment combinations basedon PD-1 pathway blockade could have even more potent effects. Clinicalevaluation of nivolumab combined with ipilimumab (anti-CTLA-4), whosemechanism of action is similar yet distinct from nivolumab's (Parry etal., 2005; Mellman et al., 2011; Topalian et al., 2012c), is ongoing andresults from a Phase 1 trial are provided herein (see, also,NCT01024231; NCT01844505; NCT01783938; Wolchok et al., 2013a; Wolchok etal., 2013b; Hodi et al., 2013). Clinical studies have also beeninitiated involving the administration of nivolumab in combination withmelanoma vaccines (NCT01176461, NCT01176474; Weber et al., 2013), withlirilumab (BMS-986015), a human IgG4 anti-KIR Ab, in patients withadvanced solid tumors (NCT01714739; Sanborn et al., 2013), withcytokines, for example, IL-21, in patients with advanced or metastaticsolid tumors (NCT01629758; Chow et al., 2013), with chemotherapeuticdrugs, for example, with platinum-based doublet chemotherapy inchemotherapy-naïve NSCLC patients (NCT01454102; Rizvi et al., 2013), andsmall-molecule targeted therapies in patients with metastatic RCC(NCT01472081; Amin et al., 2013).

In certain aspects, this disclosure relates to the combination of ananti-PD-1 Ab with different cancer treatments, includingchemotherapeutic regimes, radiation, surgery, hormone deprivation andangiogenesis inhibitors, for the treatment of various cancers. PD-1blockade may also be effectively combined with an immunogenic agent, forexample, a preparation of cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),antigen-presenting cells such as dendritic cells bearingtumor-associated antigens, cells transfected with genes encoding immunestimulating cytokines (He et al., 2004), and/or anotherimmunotherapeutic Ab (e.g., an anti-CTLA-4, anti-PD-L1 and/or anti-LAG-3Ab). Non-limiting examples of tumor vaccines that can be used includepeptides of melanoma antigens, such as peptides of gp100, MAGE antigens,Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to expressthe cytokine GM-CSF.

Combination of Anti-PD-1 and Anti-CTLA-4 Abs to Treat Advanced Melanoma

Given that immunologic checkpoints are non-redundant and can inhibitT-cell activation, proliferation and effector function within lymphnodes and/or the tumor microenvironment, and based on preclinical datathat the combination of anti-CTLA-4 and anti-PD-1 had a strongeranti-tumor effect in mouse tumor models than either Ab alone (see U.S.Pat. No. 8,008,449), the hypothesis that combined blockade of CTLA-4 andPD-1 could produce greater anti-tumor activity than single agents wastested in a clinical trial in MEL patients (Example 15).

Although not formally compared in this study, the concurrentnivolumab/ipilimumab regimen, which comprised achieved ORRs that exceedthe rates achieved with either nivolumab (Example 7) or ipilimumab alone(Hodi et al., 2010). Most importantly, rapid and deep responses wereachieved in a substantial portion of treated patients, a “deep” tumorresponse referring to a response in target lesions characterized by areduction of 80% or more from baseline measurements by radiographicassessment. In the present study, the majority of responding patients,including some with extensive and bulky tumor burden, achieved >80%tumor regression at the time of the initial tumor assessment.Particularly striking was the observation that 31% of response-evaluablepatients treated with concurrent-regimens (which comprise (i) aninduction dosing schedule with combined administration of the anti-PD-1and anti-CTLA-4 Abs followed by administration of the anti-PD-1 Abalone, and (ii) a maintenance dosing schedule comprising less frequentcombined administration of the anti-PD-1 and anti-CTLA-4 Abs)demonstrated >80% tumor regression by week 12. At the MTD for theconcurrent regimen, all 9 responding patients demonstrated >80% tumorregression with 3 CRs. In contrast, in clinical experience to date, <3%of MEL patients who received nivolumab or ipilimumab at 3 mg/kg achieveda CR (Example 7; Hodi et al., 2010). Thus, the overall activity of thisimmunotherapy combination in this preliminary phase 1 trial comparesvery favorably with that of other agents approved or being developed foradvanced melanoma, including the targeted inhibitors of activatedkinases (Chapman et al., 2011). An additional advantage of thiscombination is the durability of response as demonstrated in the presentstudy as well as longer-term immunotherapy trials with nivolumab (asdescribed herein) and ipilimumab (Wolchok et al., 2013d).

These initial data suggest that rapid responses of a greater magnitudemay be achieved in patients treated with a nivolumab/ipilimumabcombination compared with the historical experience of either agentalone. Responses were generally durable, and were observed even inpatients whose treatment was terminated early because of toxicity.Responding patients included those with elevated LDH, M1c disease andbulky multi-focal tumor burden. Similar to prior reports regardingipilimumab or nivolumab monotherapy, conventional ORRs may not fullycapture the spectrum of clinical activity and potential benefit inpatients treated with the concurrent nivolumab/ipilimumab regimen inthat a number of patients experienced either long-term SD orunconventional immune-related patterns of response. Indeed, even amongthe 7 patients in the concurrent regimen with SD ≧24 weeks or irSD ≧24weeks as best response, 6 demonstrated meaningful tumor regression of atleast 19%, and the seventh patient has declining tumor burden afterprolonged SD. Prior experience with checkpoint blockade monotherapysupports the observation that some patients may survive for extendedperiods of time with SD as the best OR, lending credence to thehypothesis that re-establishment of the equilibrium phase of immunesurveillance is a desirable outcome (Screiber et al., 2011).

The observation that patients can achieve ORs when treated sequentiallywith nivolumab after prior ipilimumab indicates that lack of response toCTLA-4 blockade does not preclude clinical benefit from PD-1 blockadeand further supports the non-redundant nature of these co-inhibitorypathways. Notably, data disclosed herein (Example 8) suggest anassociation between the occurrence of response and tumor PD-L1expression in patients receiving nivolumab, and prior data reveal acorrelation between OS and increases in peripheral ALC in patientstreated with ipilimumab (Berman et al., 2009; Ku et al., 2010; Postow etal., 2012; Delyon et al., 2013). In the present study of thenivolumab/ipilimumab combination, clinical responses were observed inpatients irrespective of lymphocyte count or baseline tumor PD-L1expression (Example 17), suggesting that the immune response generatedby combination therapy has unique features compared to eithermonotherapy, While the data suggest that baseline tumor PD-L1 expressionand lymphocyte count may be less relevant in the setting of activecombination regimens capable of inducing rapid and pronounced tumorregression, it is also notable that a different anti-PD-L1 Ab (rabbitmAb 28-8 versus mouse 5H1 mAb) was used in a different IHC assay tomeasure PD-L1 expression in the combination therapy study compared tothe nivolumab monotherapy study. In addition to changes in the IHC assayand Ab, the different results may also reflect differences in biopsysamples and tumor heterogeneity. The utility of PD-L1 expression as abiomarker for anti-PD-1 efficacy will be further evaluated prospectivelyin randomized phase 3 studies (see, e.g., NCT01721772, NCT01668784, andNCT01721746).

The spectrum of adverse events observed among patients treated with theconcurrent regimen was qualitatively similar to experience withnivolumab or ipilimumab monotherapy, although the rate of AEs wasincreased in patients treated with the combination. Grade 3-4treatment-related AEs were observed in 53% of patients treated with theconcurrent nivolumab/ipilimumab regimen, compared with historical ratesof 20% in patients treated with ipilimumab monotherapy (Hodi et al.,2010) and 17% in patients treated with nivolumab alone (Example 5) at adose of 3 mg/kg. In the sequenced-regimen cohorts, 18% of patientsexperienced grade 3-4 treatment-related AEs. AEs experienced by patientstreated with the concurrent and sequenced regimens were manageableand/or generally reversible using existing treatment algorithms.

Collectively, these results suggest that nivolumab and ipilimumab can beadministered concurrently with a manageable safety profile and leadingto durable clinical responses. More rapid and deeper clinical tumorresponses were observed in patients treated with the combinationcompared with the responses obtained with either single agent.

As of the February 2013 clinical cut-off date for the study described inExample 15, of the 52 subjects on the concurrent regimen evaluable forresponse, 21 (40%) had an OR by modified World Health Organization(mWHO) criteria (Wolchok et al., 2009). In an additional 2 subjects(4%), there was an unconfirmed OR. In Cohort 1 (0.3 mg/kg nivolumab plus3 mg/kg ipilimumab), 3 out of 14 evaluable subjects had an OR by mWHO(ORR: 21%, including 1 CR and 2 PRs). In Cohort 2 (1 mg/kg nivolumabplus 3 mg/kg ipilimumab), 9 out of 17 evaluable subjects had an OR bymWHO (ORR: 53%; including 3 CRs and 6 PRs). In Cohort 2a (3 mg/kgnivolumab plus 1 mg/kg ipilimumab), 6 out of 15 response evaluablesubjects had an OR by mWHO (ORR: 40%; including 1 CR and 5 PRs). InCohort 3 (3 mg/kg nivolumab plus 3 mg/kg ipilimumab), 3 out of 6evaluable subjects had an objective response by mWHO (ORR: 50%,including 3 PRs). Based on these data, the invention disclosed hereinincludes a method for treating a subject afflicted with a cancercomprising administering to the subject: (a) an Ab or an antigen-bindingportion thereof that specifically binds to and inhibits PD-1; and (b) anAb or an antigen-binding portion thereof that specifically binds to andinhibits CTLA-4; each Ab being administered at a dosage ranging from 0.1to 20.0 mg/kg body weight in a concurrent regimen comprising: (i) aninduction dosing schedule comprising combined administration of theanti-PD-1 and anti-CTLA-4 Abs at a dosing frequency of at least onceevery 2, 3 or 4 weeks, or at least once a month, for at least 2, 4, 6, 8or 10 doses, followed by administration of the anti-PD-1 Ab alone at adosing frequency of at least once every 2, 3 or 4 weeks, or at leastonce a month, for at least 2, 4, 6, 8 or 12 doses; followed by (ii) amaintenance dosing schedule comprising combined administration of theanti-PD-1 and anti-CTLA-4 Abs at a dosing frequency of at least onceevery 8, 12 or 16 weeks, or at least once a quarter, for at least 4, 6,8, 10, 12 or 16 doses, or for as long as clinical benefit is observed,or until unmanageable toxicity or disease progression occurs.

In certain embodiments of this method, the maintenance dosing schedulecomprises combined administration of up to 4, 6, 8, 10, 12 or 16 dosesof the anti-PD-1 and anti-CTLA-4 Abs. In other embodiments, theconcurrent regimen comprises: (i) an induction dosing schedulecomprising combined administration of the anti-PD-1 and anti-CTLA-4 Absat a dosing frequency of once every 2, 3 or 4 weeks, or once a month,for 2, 4, 6 or 8 doses, followed by administration of the anti-PD-1 Abalone at a dosing frequency of once every 2, 3 or 4 weeks, or once amonth, for 2, 4, 6, 8 or 12 doses; followed by (ii) a maintenance dosingschedule comprising combined administration of the anti-PD-1 andanti-CTLA-4 Abs at a dosing frequency of once every 8, 12 or 16 weeks,or once a quarter, for 4, 6, 8, 10, 12 or 16 doses, or for as long asclinical benefit is observed, or until unmanageable toxicity or diseaseprogression occurs. In certain other embodiments, each of the anti-PD-1and anti-CTLA-4 Abs is individually administered at a dosage of 0.1,0.3, 0.5, 1, 3, 5, 10 or 20 mg/kg. In further embodiments, the dosage ofeach of the anti-PD-1 and anti-CTLA-4 Abs is kept constant during theinduction dosing schedule and the maintenance dosing schedule. In yetother embodiments, the anti-PD-1 and anti-CTLA-4 Abs are administered atthe following dosages: (a) 0.1 mg/kg anti-PD-1 Ab and 3 mg/kg ofanti-CTLA-4 Ab; (b) 0.3 mg/kg anti-PD-1 Ab and 3 mg/kg of anti-CTLA-4Ab; (c) 1 mg/kg anti-PD-1 Ab and 3 mg/kg of anti-CTLA-4 Ab; (d) 3 mg/kganti-PD-1 Ab and 3 mg/kg of anti-CTLA-4 Ab; (e) 5 mg/kg anti-PD-1 Ab and3 mg/kg of anti-CTLA-4 Ab; (f) 10 mg/kg anti-PD-1 Ab and 3 mg/kg ofanti-CTLA-4 Ab; (g) 0.1 mg/kg anti-PD-1 Ab and 1 mg/kg of anti-CTLA-4Ab; (h) 0.3 mg/kg anti-PD-1 Ab and 1 mg/kg of anti-CTLA-4 Ab; (i) 1mg/kg anti-PD-1 Ab and 1 mg/kg of anti-CTLA-4 Ab; (j) 3 mg/kg anti-PD-1Ab and 1 mg/kg of anti-CTLA-4 Ab; (k) 5 mg/kg anti-PD-1 Ab and 1 mg/kgof anti-CTLA-4 Ab; or (l) 10 mg/kg anti-PD-1 Ab and 1 mg/kg ofanti-CTLA-4 Ab.

In the protocol described in Example 15, the 3 mg/kg nivolumab plus 3mg/kg ipilimumab dosing regimen exceeded the MTD (though combinationswith ipilimumab and other anti-PD-1 Abs may have a higher or lower MTD),whereas both Cohort 2 (1 mg/kg nivolumab plus 3 mg/kg ipilimumab) andCohort 2a (3 mg/kg nivolumab plus 1 mg/kg ipilimumab) had similarclinical activity. In addition, the majority of responses to thecombination of nivolumab and ipilimumab occurred in the first 12 weeks.Given the uncertainty of whether the ipilimumab administered past week12 contributes to the clinical benefit and the fact that the U.S. Foodand Drug Administration (FDA)- and European Medicines Agency(EMA)-approved schedule for ipilimumab is every 3 weeks for a total offour doses, in a preferred embodiment the anti-CTLA-4 Ab is administeredduring the induction dosing schedule once every 3 weeks for a total of 4doses. Monotherapy treatment with nivolumab at 3 mg/kg every two weeksuntil progression has been shown to be associated with durable responses(Examples 4-7), and a maintenance dosing schedule comprisingadministration of nivolumab every 12 weeks has been shown to beefficacious (Example 15). Thus, starting at week 12, which is after thecompletion of the four doses of combined nivolumab and ipilimumab,nivolumab at 3 mg/kg may be administered every 2 to at least 12 weeksuntil progression. Accordingly, in preferred embodiments of theconcurrent regimen method, the anti-PD-1 and anti-CTLA-4 Abs areadministered at dosages of: (a) 1 mg/kg anti-PD-1 Ab and 3 mg/kg ofanti-CTLA-4 Ab; or (b) 3 mg/kg anti-PD-1 Ab and 1 mg/kg of anti-CTLA-4Ab, and the concurrent regimen further comprises: (i) an inductiondosing schedule comprising combined administration of the anti-PD-1 andanti-CTLA-4 Abs at a dosing frequency of once every 3 weeks for 4 doses,followed by administration of the anti-PD-1 alone at a dosing frequencyof once every 3 weeks for 4 doses; followed by (ii) a maintenance dosingschedule comprising combined administration of the anti-PD-1 andanti-CTLA-4 antibodies at a dosing frequency of once every 2 to 12 ormore weeks for up to 8 doses, or for as long as clinical benefit isobserved, or until unmanageable toxicity or disease progression occurs.

Exposure-response analysis of nivolumab monotherapy across dose rangesof 1 mg/kg to 10 mg/kg reveals similar clinical activity (Example 7)while exposure-response analysis of 0.3 mg/kg, 3 mg/kg, and 10 mg/kg ofipilimumab monotherapy has demonstrated increasing activity withincrease in dose in a phase 2 trial (Wolchok et al., 2010). Therefore, adose of 3 mg/kg of ipilimumab (Cohort 2) may be more clinicallyimpactful than selection of 3 mg/kg of nivolumab (Cohort 2a). Thus, inmore preferred embodiments of the concurrent regimen method, theanti-PD-1 and anti-CTLA-4 Abs are administered at dosages of 1 mg/kganti-PD-1 Ab and 3 mg/kg of anti-CTLA-4 Ab.

In certain embodiments of the present methods, the anti-PD-1 andanti-CTLA-4 Abs are formulated for intravenous administration. Incertain other embodiments, when the anti-PD-1 and anti-CTLA-4 Abs areadministered in combination, they are administered within 30 minutes ofeach other. Either Ab may be administered first, that is, in certainembodiments, the anti-PD-1 Ab is administered before the anti-CTLA-4 Ab,whereas in other embodiments, the anti-CTLA-4 Ab is administered beforethe anti-PD-1 Ab. Typically, each Ab is administered intravenously overa period of 60 minutes. In further embodiments, the anti-PD-1 andanti-CTLA-4 Abs are administered concurrently, either admixed as asingle composition in a pharmaceutically acceptable formulation forconcurrent administration, or concurrently as separate compositions witheach Ab in a pharmaceutically acceptable formulation.

Data disclosed in Example 7 demonstrate that immunotherapy withnivolumab produced significant clinical activity in MEL patients whowere unresponsive to prior ipilimumab therapy. Accordingly, thisdisclosure provides a sequenced regimen method for treating a subjectafflicted with a cancer, the subject having previously been treated withan anti-CTLA-4 Ab, which method comprises administering to the subjectan Ab or an antigen-binding portion thereof that specifically binds toand inhibits PD-1 at a dosage ranging from 0.1 to 20.0 mg/kg body weightand at a dosing frequency of at least once every week, at least onceevery 2, 3 or 4 weeks, or at least once a month, for up to 6 to up to 72doses, or for as long as clinical benefit is observed, or untilunmanageable toxicity or disease progression occurs. In certainembodiments of this method, administration of the anti-PD-1 Ab to thesubject is initiated within 1-24 weeks after last treatment with theanti-CTLA-4 Ab. In other embodiments, administration of the anti-PD-1 Abto the subject is initiated within 1, 2, 4, 8, 12, 16, 20 or 24 weeksafter last treatment with the anti-CTLA-4 Ab. In preferred embodiments,administration of the anti-PD-1 Ab is initiated within 4, 8 or 12 weeksafter last treatment of the subject with the anti-CTLA-4 Ab. Certainembodiments of the method comprise administering the anti-PD-1 Ab at adosage of 0.1-20 mg/kg, e.g., 0.1, 0.3, 0.5, 1, 3, 5, 10 or 20 mg/kg. Inpreferred embodiments, the anti-PD-1 Ab is administered at a dosage of 1or 3 mg/kg. In certain embodiments, the sequenced regimen comprisesadministering the anti-PD-1 Ab to the subject at a dosing frequency ofonce every week, once every 2, 3 or 4 weeks, or once a month for 6 to 72doses, or for as long as clinical benefit is observed, or untilunmanageable toxicity or disease progression occurs. In preferredembodiments, the anti-PD-1 is administered at a dosage of 1 or 3 mg/kgat a dosing frequency of once every 2 weeks for up to 48 doses. In otherpreferred embodiments, the anti-PD-1 Ab is formulated for intravenousadministration.

In certain aspects of any of the present concurrent or sequenced regimenmethods, the treatment produces at least one therapeutic effect chosenfrom a reduction in size and/or growth of a tumor, elimination of thetumor, reduction in number of metastatic lesions over time, completeresponse, partial response, and stable disease. Based on the broadspectrum of cancers in which nivolumab has shown clinical response, thepresent combination therapy methods are also applicable to diversecancers. Examples of cancers that may be treated by these methodsinclude liver cancer, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, breast cancer, lung cancer, cutaneous orintraocular malignant melanoma, renal cancer, uterine cancer, ovariancancer, colorectal cancer, colon cancer, rectal cancer, cancer of theanal region, stomach cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,solid tumors of childhood, cancer of the bladder, cancer of the kidneyor ureter, carcinoma of the renal pelvis, neoplasm of the CNS, primaryCNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, environmentally induced cancers including those induced byasbestos, hematologic malignancies including, for example, multiplemyeloma, B-cell lymphoma, Hodgkin lymphoma/primary mediastinal B-celllymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronicmyelogenous leukemia, chronic lymphoid leukemia, lymphocytic lymphoma,follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides,anaplastic large cell lymphoma, T-cell lymphoma, and precursorT-lymphoblastic lymphoma, and any combinations of said cancers. Thepresent invention is also applicable to treatment of metastatic,refractory or recurring cancers. In preferred embodiments, the cancer tobe treated is chosen from MEL, RCC, squamous NSCLC, non-squamous NSCLC,CRC, CRPC, OV, GC, HCC, PC, squamous cell carcinoma of the head andneck, carcinomas of the esophagus, gastrointestinal tract and breast,and a hematological malignancy. In more preferred embodiments, thecancer is MEL.

In certain embodiments, the subject has been pre-treated for the cancer.For example, the patient may have been treated with 1 or 2 or more priorsystemic regimens of the type described herein as standard-of-caretherapeutics. In certain other embodiments, the cancer is an advanced,recurring, metastatic and/or refractory cancer. In preferredembodiments, the concurrent or sequenced regimen treatment induces adurable clinical response in the subject. In preferred embodiments, thesubject is a human, the anti-PD-1 Ab inhibits human PD-1, and theanti-CTLA-4 Ab inhibits human CTLA-4.

The anti-PD-1 Ab used in the present methods may be any therapeuticanti-PD-1 Ab of the invention. In preferred embodiments, the anti-PD-1Ab is a mAb, which may be a chimeric, humanized or human Ab. In certainembodiments, the anti-PD-1 Ab comprises the CDR1, CDR2 and CDR3 domainsin the heavy chain variable region and the CDR1, CDR2 and CDR3 domainsin the light chain variable region of 17D8, 2D3, 4H1, 5C4 (nivolumab),4A11, 7D3 or 5F4, respectively, as described and characterized in U.S.Pat. No. 8,008,449. In further embodiments, the anti-PD-1 Ab comprisesthe heavy and light chain variable regions of 17D8, 2D3, 4H1, 5C4(nivolumab), 4A11, 7D3 or 5F4, respectively. In additional embodiments,the anti-PD-1 Ab is 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3 or 5F4.In preferred embodiments, the anti-PD-1 Ab is nivolumab.

Anti-CTLA-4 antibodies of the instant invention bind to human CTLA-4 soas to disrupt the interaction of CTLA-4 with a human B7 receptor.Because the interaction of CTLA-4 with B7 transduces a signal leading toinactivation of T-cells bearing the CTLA-4 receptor, disruption of theinteraction effectively induces, enhances or prolongs the activation ofsuch T cells, thereby inducing, enhancing or prolonging an immuneresponse. Anti-CTLA-4 Abs are described in, for example, U.S. Pat. Nos.6,051,227, 7,034,121 in PCT Application Publication Nos. WO 00/37504 andWO 01/14424. An exemplary clinical anti-CTLA-4 Ab is the human mAb 10D1(now known as ipilimumab and marketed as YERVOY®) as disclosed in U.S.Pat. No. 6,984,720. In certain aspects of any of the present methods,the anti-CTLA-4 Ab is a mAb. In certain other embodiments, theanti-CTLA-4 antibody is a chimeric, humanized or human antibody. Inpreferred embodiments, the anti-CTLA-4 antibody is ipilimumab.

This disclosure also provides the use of an anti-PD-1 Ab or anantigen-binding portion thereof in combination with an anti-CTLA-4 Ab oran antigen-binding portion thereof for the preparation of aco-administered medicament for treating a subject afflicted with acancer in a concurrent regimen, wherein the anti-PD-1 and anti-CTLA-4Abs are each administered at a dosage ranging from 0.1 to 20.0 mg/kgbody weight, and further wherein the concurrent regimen comprises: (i)an induction dosing schedule comprising combined administration of theanti-PD-1 and anti-CTLA-4 Abs at a dosing frequency of at least onceevery 2, 3 or 4 weeks, or at least once a month, for at least 2, 4, 6, 8or 12 doses, followed by administration of the anti-PD-1 Ab alone at adosing frequency of at least once every 2, 3 or 4 weeks, or at leastonce a month, for at least 2, 4, 6, 8 or 10 doses; followed by (ii) amaintenance dosing schedule comprising combined administration of theanti-PD-1 and anti-CTLA-4 Abs at a dosing frequency of at least onceevery 8, 12 or 16 weeks, or at least once a quarter, for up to 4, 6, 8,10, 12 or 16 doses, or for as long as clinical benefit is observed, oruntil unmanageable toxicity or disease progression occurs. Thedisclosure provides uses of the combination of anti-PD-1 and anti-CTLA-4Abs for the preparation of co-administered medicaments corresponding toall the embodiments of the methods of treatment employing these Absdescribed herein, and are broadly applicable to the full range ofcancers disclosed herein.

The disclosure also provides an anti-PD-1 Ab or an antigen-bindingportion thereof for use in combination with an anti-CTLA-4 Ab or anantigen-binding portion thereof for treating a subject afflicted with acancer in a concurrent regimen, wherein the anti-PD-1 and anti-CTLA-4Abs are each administered at a dosage ranging from 0.1 to 20.0 mg/kgbody weight, and further wherein the concurrent regimen comprises: (i)an induction dosing schedule comprising combined administration of theanti-PD-1 and anti-CTLA-4 antibodies at a dosing frequency of at leastonce every 2, 3 or 4 weeks, or at least once a month, for at least 2, 4,6, 8 or 12 doses, followed by administration of the anti-PD-1 alone at adosing frequency of at least once every 2, 3 or 4 weeks, or at leastonce a month, for at least 2, 4, 6, 8 or 10 doses; followed by (ii) amaintenance dosing schedule comprising combined administration of theanti-PD-1 and anti-CTLA-4 antibodies at a dosing frequency of at leastonce every 8, 12 or 16 weeks, or at least once a quarter, for up to 4,6, 8, 10, 12 or 16 doses, or for as long as clinical benefit isobserved, or until unmanageable toxicity or disease progression occurs.

Methods for screening a patient population and selecting a patient assuitable for immunotherapy with the combination of anti-PD-1 andanti-CTLA-4 using the concurrent or sequenced regimens, and methods ofpredicting efficacy of the Ab combination, based on a PD-L1 biomarkerassay are performed as described for anti-PD-1 monotherapy, to theextent this biomarker is applicable to combination therapy withanti-PD-1 and anti-CTLA-4 Abs.

The disclosure additionally provides a kit for treating a subjectafflicted with a cancer, the kit comprising: (a) a dosage ranging from0.1 to 20.0 mg/kg body weight of an Ab or an antigen-binding portionthereof that specifically binds to and inhibits PD-1; (b) a dosageranging from 0.1 to 20.0 mg/kg of an Ab or an antigen-binding portionthereof that specifically binds to and inhibits CTLA-4; and (c)instructions for using the combination of the anti-PD-1 and anti-CTLA-4antibodies in any of the concurrent regimen methods. In certainembodiments, some of the dosages of the anti-PD-1 and anti-CTLA-4 Absare admixed within a single pharmaceutical formulation for concurrentadministration. In other embodiments, the dosages of the anti-PD-1 andanti-CTLA-4 Abs are formulated as separate compositions with each Ab ina pharmaceutically acceptable formulation.

The disclosure further provides a kit for treating a subject afflictedwith a cancer, the kit comprising: (a) a dosage ranging from 0.1 to 20.0mg/kg body weight of an Ab or an antigen-binding portion thereof thatspecifically binds to and inhibits PD-1; and (b) instructions for usingthe anti-PD-1 Ab in any of the sequenced regimen methods.

A combined PD-1 and CTLA-4 blockade may also be further combined withstandard cancer treatments. For example, a combined PD-1 and CTLA-4blockade may be effectively combined with chemotherapeutic regimes, forexample, further combination with dacarbazine or IL-2, for the treatmentof MEL. In these instances, it may be possible to reduce the dose of thechemotherapeutic reagent. The scientific rationale behind the combineduse of PD-1 and CTLA-4 blockade with chemotherapy is that cell death,which is a consequence of the cytotoxic action of most chemotherapeuticcompounds, should result in increased levels of tumor antigen in theantigen presentation pathway. Other combination therapies that mayresult in synergy with a combined PD-1 and CTLA-4 blockade through celldeath include radiation, surgery, or hormone deprivation. Each of theseprotocols creates a source of tumor antigen in the host. Angiogenesisinhibitors may also be combined with a combined PD-1 and CTLA-4blockade. Inhibition of angiogenesis leads to tumor cell death, whichmay also be a source of tumor antigen to be fed into host antigenpresentation pathways.

Immunotherapy of Cancer Patients Using an Anti-PD-L1 Antibody

PD-L1 is the primary PD-1 ligand up-regulated within solid tumors, whereit can inhibit cytokine production and the cytolytic activity ofPD-1-positive, tumor-infiltrating CD4⁺ and CD8⁺ T-cells, respectively(Dong et al., 2002; Hino et al., 2010; Taube et al., 2012). Theseproperties make PD-L1 a promising target for cancer immunotherapy. Theclinical trials of anti-PD-L1 immunotherapy described in the Examplesdemonstrate for the first time that mAb blockade of the immuneinhibitory ligand, PD-L1, produces both durable tumor regression andprolonged (≧24 weeks) disease stabilization in patients with metastaticNSCLC, MEL, RCC and OV, including those with extensive prior therapy.The human anti-PD-L1 HuMAb, BMS-936559, had a favorable safety profileoverall at doses up to and including 10 mg/kg, as is evident from thelow (9%) incidence of grade 3-4 drug-related AEs. These findings areconsistent with the mild autoimmune phenotype seen in PD-L1^(−/−) mice(Dong et al., 2004) and the more severe hyperproliferation seen inCTLA-4^(−/−) mice relative to PD-1^(−/−) mice (Phan et al., 2003; Tivolet al., 1995; Nishimura et al., 1999). Most of the toxicities associatedwith anti-PD-L1 administration in patients were immune-related,suggesting on-target effects. The spectrum and frequency of adverseevents of special interest (AEOSIs) is somewhat different betweenanti-PD-L1 and anti-CTLA-4, emphasizing the distinct biology of thesepathways (Ribas et al., 2005). Infusion reactions were observed withBMS-936559, although they were mild in most patients. Severe colitis, adrug-related AE observed in ipilimumab-treated patients (Beck et al.,2006), was infrequently noted with anti-PD-L1.

As noted above for anti-PD-1 immunotherapy, another important feature ofanti-PD-L1 therapy is the durability of responses across multiple tumortypes. This is particularly notable considering the advanced disease andprior treatment of patients on the current study. Although not compareddirectly, this durability appears greater than that observed with mostchemotherapies and kinase inhibitors used to treat these cancers.

Because peripheral blood T-cells express PD-L1, it is possible to assessin vivo RO by BMS-963559 as a pharmacodynamic measure. Median RO was65.8%, 66.2%, and 72.4% for the doses tested. Whereas these studiesprovide a direct assessment and evidence of target engagement inpatients treated with BMS-936559, relationships between RO in peripheralblood and the tumor microenvironment remain poorly understood.

Based on the clinical data disclosed herein, this disclosure provides amethod for immunotherapy of a subject afflicted with cancer, whichmethod comprises administering to the subject a composition comprising atherapeutically effective amount of an anti-PD-L1 Ab of the invention oran antigen-binding portion thereof. The disclosure also provides amethod of inhibiting growth of tumor cells in a subject, comprisingadministering to the subject an anti-PD-L1 Ab of the invention or anantigen-binding portion thereof. In preferred embodiments, the subjectis a human. In certain embodiments, the Ab or antigen-binding portionthereof is of an IgG1 or IgG4 isotype. In other embodiments, the Ab orantigen-binding portion thereof is a mAb or an antigen-binding portionthereof. In further embodiments, the Ab or antigen-binding portionthereof is a chimeric, humanized or human Ab or an antigen-bindingportion thereof. In preferred embodiments for treating a human patient,the Ab or antigen-binding portion thereof is a human Ab or anantigen-binding portion thereof.

Clinical trials described in the Examples employed the anti-PD-L1 HuMAbBMS-936559 to treat cancer. While BMS-936559 (designated HuMAb 12A4 inU.S. Pat. No. 7,943,743) was selected as the lead anti-PD-L1 Ab forentering the clinic, it is notable that several anti-PD-L1 Abs of theinvention share with 12A4 functional properties that are important tothe therapeutic activity of 12A4, including high affinity bindingspecifically to human PD-L1, increasing T-cell proliferation, IL-2secretion and interferon-γ production in an MLR assay, inhibiting thebinding of PD-L1 to PD-1, and reversing the suppressive effect of Tregulatory cells on T cell effector cells and/or dendritic cells.Moreover, certain of the anti-PD-L1 Abs of the invention, namely 1B12,7H1 and 12B7 are structurally related to 12A4 in comprising V_(H) andV_(κ) regions that have sequences derived from V_(H) 1-69 and V_(κ) L6germline sequences, respectively. In addition, at least 12B7, 3G10, 1B12and 13G4 cross-compete with 12A4 for binding to the same epitope regionof hPD-L1, whereas 5F8 and 10A5 may bind to the same or an overlappingepitope region as 12A4 (Examples 2 and 3). Thus, the preclinicalcharacterization of 12A4 and other anti-PD-L1 HuMabs indicate that themethods of treating cancer provided herein may be performed using any ofthe broad genus of anti-PD-L1 Abs of the invention.

Accordingly, this disclosure provides immunotherapy methods comprisingadministering to a patient an anti-PD-L1 Ab or antigen-binding portionthereof comprising (a) a heavy chain variable region that comprisesconsecutively linked amino acids having a sequence derived from a humanV_(H) 1-18 germline sequence, and a light chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(κ) L6 germline sequence; (b) a heavy chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(H) 1-69 germline sequence, and a light chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(κ) L6 germline sequence; (c) a heavychain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(H) 1-3 germline sequence, anda light chain variable region that comprises consecutively linked aminoacids having a sequence derived from a human V_(κ) L15 germlinesequence; (d) a heavy chain variable region that comprises consecutivelylinked amino acids having a sequence derived from a human V_(H) 1-69germline sequence, and a light chain variable region that comprisesconsecutively linked amino acids having a sequence derived from a humanV_(κ) A27 germline sequence; (e) a heavy chain variable region thatcomprises consecutively linked amino acids having a sequence derivedfrom a human V_(H) 3-9 germline sequence, and a light chain variableregion that comprises consecutively linked amino acids having a sequencederived from a human V_(κ) L15germline sequence; or (f) a heavy chainvariable region that comprises consecutively linked amino acids having asequence derived from a human V_(H) 3-9 germline sequence, and a lightchain variable region that comprises consecutively linked amino acidshaving a sequence derived from a human V_(κ) L18 germline sequence. Incertain embodiments, the anti-PD-L1 Ab or antigen-binding portionthereof administered to the patient cross-competes for binding to PD-L1with a reference Ab or a reference antigen-binding portion thereofcomprising: (a) a human heavy chain variable region comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 15 and a human light chain variable region comprising consecutivelylinked amino acids having the sequence set forth in SEQ ID NO: 25; (b) ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 16 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 26; (c) a human heavy chainvariable region comprising consecutively linked amino acids having thesequence set forth in SEQ ID NO: 17 and a human light chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34. In preferred embodiments, the Ab orantigen-binding portion thereof cross-competes for binding to PD-1 witha reference Ab or reference antigen-binding portion thereof comprising ahuman heavy chain variable region comprising consecutively linked aminoacids having the sequence set forth in SEQ ID NO: 16 and a human lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO: 26.

In certain preferred embodiments of the immunotherapy methods disclosedherein, the anti-PD-L1 Ab or antigen-binding portion thereofadministered to the subject comprises: (a) a human heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 15 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 25; (b) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 16 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 26; (c) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 17 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 27; (d) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 18 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 28; (e) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 19 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 29; (f) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 20 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 30; (g) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 21 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 31; (h) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 22 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 32; (i) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 23 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 33; or (j) a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 24 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 34. In more preferred embodiments, the anti-PD-L1 Abor antigen-binding portion comprises a human heavy chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 16 and a human light chain variable regioncomprising consecutively linked amino acids having the sequence setforth in SEQ ID NO: 26.

Broad Spectrum of Cancers Treatable by Anti-PD-L1 Immunotherapy

The clinical activity of anti-PD-L1 in patients with advanced NSCLC,similar to the activity of anti-PD-1 in these patients, was surprisingand unexpected since NSCLC has been considered to be poorly responsiveto immune-based therapies (Holt and Disis, 2008; Holt et al., 2011). Thepresent clinical data obtained with BMS-936559, an anti-PD-L1 Ab of theinvention, substantiate and extend the evidence obtained using theanti-PD-1 Ab that immunotherapy based on PD-1 blockade is not applicableonly to “immunogenic” tumor types, such as MEL and RCC, but is alsoeffective with a broad range of cancers, including treatment-refractorymetastatic NSCLC, that are generally not considered to beimmune-responsive. Preferred cancers that may be treated using theanti-PD-L1 Abs of the invention include MEL (e.g., metastatic malignantmelanoma), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer(OV), gastric cancer (GC), breast cancer (BC), pancreatic carcinoma (PC)and carcinoma of the esophagus. Additionally, the invention includesrefractory or recurrent malignancies whose growth may be inhibited usingthe anti-PD-L1 Abs of the invention.

Accordingly, examples of cancers that may be treated using an anti-PD-L1Ab in the methods of the invention, based on the indications of verybroad applicability of anti-PD-L1 immunotherapy provided herein, includebone cancer, skin cancer, cancer of the head or neck, breast cancer,lung cancer, cutaneous or intraocular malignant melanoma, renal cancer,uterine cancer, castration-resistant prostate cancer, colon cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, carcinomas of the ovary, gastrointestinal tractand breast, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma, multiplemyeloma, environmentally induced cancers including those induced byasbestos, metastatic cancers, and any combinations of said cancers. Thepresent invention is also applicable to treatment of metastatic cancers.

Combination Therapy with Anti-PD-L1 Abs

Optionally, Abs to PD-L1 can be combined with an immunogenic agent, forexample a preparation of cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),antigen-presenting cells such as dendritic cells bearingtumor-associated antigens, and cells transfected with genes encodingimmune stimulating cytokines (He et al., 2004). Non-limiting examples oftumor vaccines that can be used include peptides of melanoma antigens,such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF.PD-L1 blockade may also be effectively combined with standard cancertreatments, including chemotherapeutic regimes, radiation, surgery,hormone deprivation and angiogenesis inhibitors, as well as anotherimmunotherapeutic Ab (e.g., an anti-PD-1, anti-CTLA-4 or anti-LAG-3 Ab).

Uses of Anti-PD-L1 Abs

This disclosure provides the use of any anti-PD-L1 Ab or antigen-bindingportion thereof of the invention for the preparation of a medicament forinhibiting signaling from the PD-1/PD-L1 pathway so as to therebypotentiate an endogenous immune response in a subject afflicted withcancer. This disclosure also provides the use of any anti-PD-L1 Ab orantigen-binding portion thereof of the invention for the preparation ofa medicament for immunotherapy of a subject afflicted with cancercomprising disrupting the interaction between PD-1 and PD-L1. Thedisclosure provides medical uses of any anti-PD-L1 Ab or antigen-bindingportion thereof of the invention corresponding to all the embodiments ofthe methods of treatment employing an anti-PD-L1 Ab described herein.

This disclosure also provides an anti-PD-L1 Ab or an antigen-bindingportion thereof of the invention for use in treating a subject afflictedwith cancer comprising potentiating an endogenous immune response in asubject afflicted with cancer by inhibiting signaling from thePD-1/PD-L1 pathway. The disclosure further provides an anti-PD-L1 Ab oran antigen-binding portion thereof of the invention for use inimmunotherapy of a subject afflicted with cancer comprising disruptingthe interaction between PD-1 and PD-L1. These Abs may be used inpotentiating an endogenous immune response against, or in immunotherapyof, the full range of cancers disclosed herein. In preferredembodiments, the cancers include MEL (e.g., metastatic malignant MEL),RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer (OV),gastric cancer (GC), breast cancer (BC), pancreatic carcinoma (PC) andcarcinoma of the esophagus.

Validation of Cancer Immunotherapy by Immune Checkpoint Blockade

A major implication of the clinical activity of immune checkpointblockade is that significant endogenous immune responses to tumorantigens are generated and these responses may be harnessedtherapeutically to mediate clinical tumor regression upon checkpointinhibition. In fact, there is evidence that inhibitory ligands such asPD-L1 are induced in response to immune attack, a mechanism termedadaptive resistance (Gajewski et al., 2010; Taube et al., 2012). Thispotential mechanism of immune resistance by tumors suggests thatPD-1/PD-L1-directed therapy might synergize with other treatments thatenhance endogenous antitumor immunity. Follow-up studies have verifiedthat patients continue to demonstrate tumor control after cessation ofPD-1/PD-L1 pathway blockade (Lipson et al., 2013). Such tumor controlmay reflect a persistent antitumor immune response and the generation ofeffective immunologic memory to enable sustained control of tumorgrowth.

The data disclosed herein on the clinical testing of Abs that block theimmunoregulatory receptor, PD-1, and also of Abs that block one of itscognate ligands, PD-L1, are unprecedented. These data constitute thelargest clinical experience to date with PD-1 pathway-directed cancerimmunotherapy, and the first report specifically describing the safety,tolerability, and initial clinical activity of an anti-PD-L1-directedagent. These findings show that both anti-PD-1 and anti-PD-L1 havefavorable overall safety profiles and provide clear evidence of clinicalactivity across diverse cancers, including NSCLC, a tumor nothistorically considered responsive to immunotherapy, as well as tumorsknown to respond to immunotherapy, including MEL, RCC and OV. Thus,these data strongly validate the PD-1/PD-L1 pathway as an importanttarget for therapeutic intervention in cancer.

The remarkable similarities observed between the patterns of clinicalactivity obtained with the anti-PD-1 and anti-PD-L1 mAbs, and among thetumor types analyzed to date, validate the general importance of thePD-1/PD-L1 signaling pathway in tumor immune resistance and as a targetfor therapeutic intervention. Although the molecular interactionsblocked by these two Abs are not identical, it has been clearlydemonstrated herein that, irrespective of mechanistic details, bothanti-PD-1 and anti-PD-L1 Abs of the invention are effective in treatingpatients afflicted with a wide variety of cancers.

Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. For example, another aspectof this disclosure provides a method of treating an infectious diseasein a subject comprising administering to the subject an anti-PD1 or ananti-PD-L1 Ab, or antigen-binding portion thereof, of the invention suchthat the subject is treated for the infectious disease. Preferably, theAb is a human anti-human PD-1 or PD-L1 Ab (such as any of the human Absdescribed herein). Alternatively, the Ab is a chimeric or humanized Ab.

Similar to its application to tumors as discussed above, Ab-mediatedPD-1 or PD-L1 blockade can be used alone, or as an adjuvant, incombination with vaccines, to potentiate an immune response topathogens, toxins, and/or self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, and C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. PD-1 and/or PD-L1 blockade is particularly useful againstestablished infections by agents such as HIV that present alteredantigens over the course of an infection. Novel epitopes on theseantigens are recognized as foreign at the time of anti-human PD-1 orPD-L1 administration, thus provoking a strong T cell response that isnot dampened by negative signals through the PD-1/PD-L1 pathway.

In the above methods, PD-1 or PD-L1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., administrationof interferons, GM-CSF, G-CSF or IL-2).

Kits

Also within the scope of the present invention are kits, includingpharmaceutical kits, comprising an anti-PD-1 and/or an anti-PD-L1 Ab ofthe invention, or a combination of an anti-PD-1 and an anti-CTLA-4 Ab,for therapeutic uses, and diagnostic kits comprising an anti-PD-L1 Ab ofthe invention for assaying membranous PD-L1 expression as a biomarkerfor screening patients for immunotherapy or for predicting the efficacyof an immunotherapeutic agent. Kits typically include a label indicatingthe intended use of the contents of the kit and instructions for use.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit. In certainembodiments of a pharmaceutical kit, the anti-PD-1 and/or anti-PD-L1 Absmay be co-packaged with other therapeutic agents in unit dosage form. Incertain embodiments of a diagnostic kit, the anti-PD-L1 Ab may beco-packaged with other reagents for performing an assay to detect and/orquantify PD-L1 expression.

In certain preferred embodiments, the pharmaceutical kit comprises theanti-human PD-1 HuMAb, nivolumab. In other preferred embodiments, thepharmaceutical kit comprises the anti-human PD-L1 HuMAb, BMS-936559. Inyet other preferred embodiments, the pharmaceutical kit comprises theanti-human CTLA-4 HuMAb, ipilimumab. In certain preferred embodiments,the diagnostic kit comprises the rabbit anti-human PD-L1 mAb, 28-8,comprising the V_(H) and V_(κ) regions whose amino acid sequences areset forth in SEQ ID NOs. 35 and 36, respectively. In other preferredembodiments, the diagnostic kit comprises the murine anti-human PD-L1mAb, 5H1 (Dong et al., 2002).

PD-L1 Biomarker for Predicting Anti-PD-1 Efficacy

A particular challenge in cancer immunotherapy has been theidentification of mechanism-based predictive biomarkers to enablepatient selection and guide on-treatment management. Data disclosed inthe Examples below indicate that cell surface PD-L1 expression in tumorsis a useful molecular marker for predicting the efficacy of, andselecting patients for, immunotherapy with anti-PD-1 and potentiallyother immune checkpoint inhibitors.

There are conflicting reports in the literature about the clinicalimplications of PD-L1 being expressed in tumors. Several studies haveconcluded that PD-L1 expression in tumors correlates with a poorprognosis for the patient. See, e.g., Hino et al. (2010) (MEL);Hamanishi et al. (2007) (OV); Thompson et al. (2006) (RCC). Thesefindings may be rationalized on the basis that the interaction of PD-L1on tumor cells and PD-1 on T cells helps abrogate immune responsesdirected against the tumor, resulting in immune evasion fromtumor-specific T cells (Blank et al., 2005). However, in contrast to theforegoing studies, Gadiot et al. (2011) and Taube et al. (2012) haverecently reported that PD-L1 expression in melanoma tumors correlateswith a trend toward better survival. These seemingly contradictory datamay reflect the relatively small numbers of patients analyzed, differenthistologic subtypes studied, or different methodologies used, e.g., theuse of different Abs to stain PD-L1, the use of frozen versusparaffin-embedded material for IHC, and the detection of membranousand/or cytoplasmic staining of PD-L1. Taube et al. (2012) note thatPD-L1 is a type I transmembrane molecule, and hypothesize that while thecytoplasmic presence of PD-L1 may represent intracellular stores of thispolypeptide that may be deployed to the cell surface upon appropriatestimulation, it is cell surface PD-L1 expression of that is biologicallyrelevant as a potential biomarker for predicting clinical response toPD-1 blockade. See, also, Brahmer et al. (2010), which describespreliminary evidence, obtained on a small sample size of only 9patients, of a correlation between membranous PD-L1 expression andanti-PD-1 efficacy. The data described in the Examples below on the useof membranous PD-L1 expression as a biomarker for anti-PD-1 efficacy,which was obtained from analysis of a much larger sample, substantiatethe hypothesis that PD-L1 expression may be used as a biomarker forpredicting anti-PD-1 clinical response and for screening patients toidentify suitable candidates for immunotherapy with an anti-PD-1 Ab.Though the utility of this biomarker was demonstrated for screeningpatients for, or predicting the clinical response to, anti-PD-1immunotherapy, PD-L1 expression may also potentially be applicable morebroadly as a companion biomarker for other types of inhibitors ofinhibitory immunoregulators.

Specifically, membranous PD-L1 expression was assayed using an automatedIHC protocol and a rabbit anti-hPD-L1 Ab. Strikingly, in the initial setof data analyzed (see Example 8), no patients with cell surfacePD-L1-negative tumors (MEL, NSCLC, CRC, RCC and CRPC) experienced an ORfollowing treatment with the anti-PD-1 Ab, nivolumab. In contrast, cellsurface expression of PD-L1 on tumor cells in pretreatment biopsies maybe associated with an increased rate of OR among patients treated withnivolumab. While tumor cell expression of PD-L1 may be driven byconstitutive oncogenic pathways, it may also reflect “adaptive immuneresistance” in response to an endogenous antitumor immune response, partof a host inflammatory response, which may remain in check unlessunleashed by blockade of the PD-1/PD-L1 pathway (Taube et al., 2012).This emerging concept of adaptive immune resistance in cancer immunologysuggests that inhibitory ligands such as PD-L1 are induced in responseto immune attack (Gajewski et al., 2010; Taube et al., 2012). A majorimplication of the clinical activity of immune checkpoint blockade asdescribed herein is that significant endogenous immune responses totumor antigens are generated and these responses may be harnessedtherapeutically to mediate clinical tumor regression upon checkpointinhibition. This potential mechanism of immune resistance by tumorssuggests that PD-1/PD-L1-directed therapy might synergize with othertreatments that enhance endogenous antitumor immunity.

Assaying Cell-Surface PD-L1 Expression by Automated IHC

As described in the Examples, an automated IHC method was developed forassaying the expression of PD-L1 on the surface of cells in FFPE tissuespecimens. This disclosure provides methods for detecting the presenceof human PD-L1 antigen in a test tissue sample, or quantifying the levelof human PD-L1 antigen or the proportion of cells in the sample thatexpress the antigen, which methods comprise contacting the test sample,and a negative control sample, with a mAb that specifically binds tohuman PD-L1, under conditions that allow for formation of a complexbetween the Ab or portion thereof and human PD-L1. Preferably, the testand control tissue samples are FFPE samples. The formation of a complexis then detected, wherein a difference in complex formation between thetest sample and the negative control sample is indicative of thepresence of human PD-L1 antigen in the sample. Various methods are usedto quantify PD-L1 expression.

In a particular embodiment, the automated IHC method comprises: (a)deparaffinizing and rehydrating mounted tissue sections in anautostainer; (b) retrieving antigen using a decloaking chamber and pH 6buffer, heated to 110° C. for 10 min; (c) setting up reagents on anautostainer; and (d) running the autostainer to include steps ofneutralizing endogenous peroxidase in the tissue specimen; blockingnon-specific protein-binding sites on the slides; incubating the slideswith primary Ab; incubating with a post-primary blocking agent;incubating with NovoLink Polymer; adding a chromogen substrate anddeveloping; and counterstaining with hematoxylin.

For assessing PD-L1 expression in tumor tissue samples, a pathologistexamines the number of membrane PD-L1⁺ tumor cells in each field under amicroscope and mentally estimates the percentage of cells that arepositive, then averages them to come to the final percentage. Thedifferent staining intensities are defined as 0/negative, 1+/weak,2+/moderate, and 3+/strong. Typically, percentage values are firstassigned to the 0 and 3+ buckets, and then the intermediate 1+ and 2+intensities are considered. For highly heterogeneous tissues, thespecimen is divided into zones, and each zone is scored separately andthen combined into a single set of percentage values. The percentages ofnegative and positive cells for the different staining intensities aredetermined from each area and a median value is given to each zone. Afinal percentage value is given to the tissue for each stainingintensity category: negative, 1+, 2+, and 3+. The sum of all stainingintensities needs to be 100%.

Staining is also assessed in tumor-infiltrating inflammatory cells suchas macrophages and lymphocytes. In most cases macrophages serve as aninternal positive control since staining is observed in a largeproportion of macrophages. While not required to stain with 3+intensity, an absence of staining of macrophages should be taken intoaccount to rule out any technical failure. Macrophages and lymphocytesare assessed for plasma membrane staining and only recorded for allsamples as being positive or negative for each cell category Staining isalso characterized according to an outside/inside tumor immune celldesignation. “Inside” means the immune cell is within the tumor tissueand/or on the boundaries of the tumor region without being physicallyintercalated among the tumor cells. “Outside” means that there is nophysical association with the tumor, the immune cells being found in theperiphery associated with connective or any associated adjacent tissue.

In certain embodiments of these scoring methods, the samples are scoredby two pathologists operating independently and the scores aresubsequently consolidated. In certain other embodiments, theidentification of positive and negative cells is scored usingappropriate software.

A histoscore is used as a more quantitative measure of the IHC data. Thehistoscore is calculated as follows:

Histoscore=[(% tumor×1 (low intensity))+(% tumor×2 (mediumintensity))+(% tumor×3 (high intensity)]

To determine the histoscore, the pathologist estimates the percentage ofstained cells in each intensity category within a specimen. Becauseexpression of most biomarkers is heterogeneous the histoscore is a truerrepresentation of the overall expression. The final histoscore range is0 (no expression) to 300 (maximum expression).

An alternative means of quantifying PD-L1 expression in a test tissuesample IHC is to determine the adjusted inflammation score (AIS) scoredefined as the density of inflammation multiplied by the percent PD-L1expression by tumor-infiltrating inflammatory cells (Taube et al.,2012).

Cancer Immunotherapy with Anti-PD-1 Comprising a Patient Selection Step

This disclosure also provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is a suitable candidate for immunotherapy, e.g., administration ofan anti-PD-1 Ab, the selecting comprising (i) optionally providing atest tissue sample obtained from a patient with cancer of the tissue,the test tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells, (ii) assessing the proportion of cells in the testtissue sample that express PD-L1 on the cell surface, and (iii)selecting the subject as a suitable candidate based on an assessmentthat the proportion of cells in the test tissue sample that expressPD-L1 on the cell surface exceeds a predetermined threshold level; and(b) administering to the selected subject a composition comprising atherapeutically effective amount of an agent that inhibits signalingfrom an inhibitory immunoregulator e.g., an anti-PD-1 Ab.

There is evidence that membranous PD-L1 expression is a surrogate for anendogenous antitumor immune response that is part of a host inflammatoryresponse (Gajewski et al., 2010; Taube et al., 2012). Accordingly, cellsurface expression of PD-L1 in tumors and/or inflammatory cells in thetumor microenvironment may be a marker not just for selecting cancerpatients who would benefit from treatment with an anti-PD-1 Ab, but alsofor treatment with an anti-PD-L1 Ab as well as treatments targetinginhibitory immunoregulatory pathways other than the PD-1/PD-L1 pathway.For example, cell surface expression of PD-L1 in tumors and/ortumor-infiltrating inflammatory cells may be used as a marker foridentifying or selecting suitable cancer patients who would benefit fromimmunotherapy with agents, including Abs, that target, and disrupt orinhibit signaling from, immune checkpoints such as PD-L1, CytotoxicT-Lymphocyte Antigen-4 (CTLA-4), B and T Lymphocyte Attenuator (BTLA), Tcell Immunoglobulin and Mucin domain-3 (TIM-3), Lymphocyte ActivationGene-3 (LAG-3), Killer Immunoglobulin-like Receptor (KIR), Killer cellLectin-like Receptor G1 (KLRG-1), Natural Killer Cell Receptor 2B4(CD244), and CD160 (Pardoll, 2012; Baitsch et al., 2012). In certainpreferred embodiments, the inhibitory immunoregulator is a component ofthe PD-1/PD-L1 signaling pathway. In other preferred embodiments, theinhibitory immunoregulator is an anti-PD-1 Ab of the invention. In yetother preferred embodiments, the inhibitory immunoregulator is ananti-PD-L1 Ab of the invention.

Where any immunotherapy methods comprising assaying PD-L1 expression,i.e., employing a PD-L1 expression biomarker, are described below ascomprising the selection of a patient who is, or is not, suitable foranti-PD-1 immunotherapy, or as comprising the administration of ananti-PD-1 Ab for immunotherapeutic purposes, it should be understoodthat these methods apply more broadly to the selection of a patient whois, or is not, suitable for immunotherapy with, or to the administrationof an inhibitor of, an inhibitory immunoregulator (e.g., CTLA-4, BTLA,TIM3, LAG3 or KIR) or a component or ligand thereof. Further, in any themethods comprising the measurement of PD-L1 expression in a test tissuesample, it should be understood that the step comprising the provisionof a test tissue sample obtained from a patient is an optional step.That is, in certain embodiments the method includes this step, and inother embodiments, this step is not included in the method. It shouldalso be understood that in certain preferred embodiments the “assessing”step to identify, or determine the number or proportion of, cells in thetest tissue sample that express PD-L1 on the cell surface is performedby a transformative method of assaying for PD-L1 expression, for exampleby performing a reverse transcriptase-polymerase chain reaction (RT-PCR)assay or an IHC assay. In certain other embodiments, no transformativestep is involved and PD-L1 expression is assessed by, for example,reviewing a report of test results from a laboratory. In certainembodiments, the steps of the methods up to, and including, assessingPD-L1 expression provides an intermediate result that may be provided toa physician or other medical practitioner for use in selecting asuitable candidate for immunotherapy and/or administering animmunotherapeutic agent to the patient. In certain embodiments, thesteps that provide the intermediate result may be performed by a medicalpractitioner or someone acting under the direction of a medicalpractitioner. In other embodiments, these steps are performed by anindependent laboratory or by an independent person such as a laboratorytechnician.

The disclosure also provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) selecting a subjectthat is not suitable for treatment with an agent that inhibits aninhibitory immunoregulator, e.g., anti-PD-1 Ab immunotherapy, theselecting comprising (i) optionally providing a test tissue sampleobtained from a patient with cancer of the tissue, the test tissuesample comprising tumor cells and tumor-infiltrating inflammatory cells;(ii) assessing the proportion of cells in the test tissue sample thatexpress PD-L1 on the surface of the cells; and (iii) selecting thesubject as not suitable for immunotherapy with an inhibitor of aninhibitory immunoregulator, e.g., an anti-PD-1 Ab, based on anassessment that the proportion of cells in the test tissue sample thatexpress PD-L1 on the cell surface is less than a predetermined thresholdlevel; and (b) administering a standard-of-care therapeutic other thanan inhibitor of an inhibitory immunoregulator, e.g., an anti-PD-1 Ab, tothe selected subject.

Measurement of PD-L1 Expression

In certain embodiments of any of the present methods, the proportion ofcells that express PD-L1 is assessed by performing an assay to determinethe presence of PD-L1 RNA. In further embodiments, the presence of PD-L1RNA is determined by RT-PCR, in situ hybridization or RNase protection.In other embodiments, the proportion of cells that express PD-L1 isassessed by performing an assay to determine the presence of PD-L1polypeptide. In further embodiments, the presence of PD-L1 polypeptideis determined by immunohistochemistry (IHC), enzyme-linked immunosorbentassay (ELISA), in vivo imaging, or flow cytometry. In preferredembodiments, PD-L1 expression is assayed by IHC. Flow cytometry may beparticularly suitable for assaying PD-L1 expression in cells ofhematologic tumors. In preferred embodiments of all of these methods,cell surface expression of PD-L1 is assayed using, e.g., IHC or in vivoimaging.

Imaging techniques have provided important tools in cancer research andtreatment. Recent developments in molecular imaging systems, includingpositron emission tomography (PET), single-photon emission computedtomography (SPECT), fluorescence reflectance imaging (FRI),fluorescence-mediated tomography (FMT), bioluminescence imaging (BLI),laser-scanning confocal microscopy (LSCM) and multiphoton microscopy(MPM), will likely herald even greater use of these techniques in cancerresearch. Some of these molecular imaging systems allow clinicians tonot only see where a tumor is located in the body, but also to visualizethe expression and activity of specific molecules, cells, and biologicalprocesses that influence tumor behavior and/or responsiveness totherapeutic drugs (Condeelis and Weissleder, 2010). Ab specificity,coupled with the sensitivity and resolution of PET, makes immunoPETimaging particularly attractive for monitoring and assaying expressionof antigens in tissue samples (McCabe and Wu, 2010; Olafsen et al.,2010). In certain embodiments of any of the present methods, PD-L1expression is assayed by immunoPET imaging.

In certain embodiments of any of the present methods, the proportion ofcells in a test tissue sample that express PD-L1 is assessed byperforming an assay to determine the presence of PD-L1 polypeptide onthe surface of cells in the test tissue sample. In certain embodiments,the test tissue sample is a FFPE tissue sample. In certain preferredembodiments, the presence of PD-L1 polypeptide is determined by IHCassay. In further embodiments, the IHC assay is performed using anautomated process. In further embodiments, the IHC assay is performedusing an anti-PD-L1 mAb to bind to the PD-L1 polypeptide.

Abs that Bind Specifically to Cell-Surface-Expressed PD-L1 in FFPETissues

An Ab may bind to an antigen in fresh tissues but completely fail torecognize the antigen in an FFPE tissue sample. This phenomenon, wellknown in the art, is thought to be due primarily to intra- andinter-molecular cross-linking of polypeptides induced by formalinfixation, which alters the epitope recognized by the Ab (Sompuram etal., 2006). In addition, several factors known to influence staining inFFPE tissue, including variable time to fixation, inadequate fixationperiod, differences in fixative used, tissue processing, Ab clone anddilution, antigen retrieval, detection system, and interpretation ofresults using different threshold points are important variables thatcan affect tissue antigenicity and IHC measurements (Bordeaux et al.,2010). In particular, a lack of anti-human PD-L1 Abs that stain PD-L1 inFFPE specimens has been noted in the art (Hamanishi et al., 2007). Taubeet al. (2012) and Gadiot et al. (2011) have also reported difficultiesin identifying anti-PD-1 Abs that bind specifically to PD-L1 in FFPEtissues, and inconsistent test results on the same Abs. Our own analysisof five commercially available anti-hPD-L1 Abs shows that these Absfailed to distinguish FFPE cells expressing PD-L1 from cells that didnot express PD-L1 (see Example 9, Table 7). Thus, the contradictoryresults reported by different groups on the implications of PD-L1expression for prognosis of a tumor may, in part, reflect thedifferential abilities of anti-PD-L1 Abs used to detect PD-L1polypeptide in FFPE tissue samples. Accordingly, in order to detecthPD-L1 on the surface of cells using an IHC assay on FFPE tissues, thereis a need for anti-hPD-L1 Abs that bind specifically to cellsurface-expressed PD-L1 in FFPE tissue samples.

This disclosure provides a mAb or an antigen-binding portion thereofthat binds specifically to a cell surface-expressed PD-L1 antigen in aFFPE tissue sample. In preferred embodiments, the mAb or antigen-bindingportion thereof does not bind to a cytoplasmic PD-L1 polypeptide in theFFPE tissue sample or exhibits a very low level of background binding.In certain other embodiments, the presence or absence of bindingspecifically to a cell surface-expressed or a cytoplasmic PD-L1polypeptide is detected by immunohistochemical staining. In certainpreferred aspects of the invention, the mAb or antigen-binding portionis a rabbit Ab or a portion thereof. In other preferred embodiments, themAb is the rabbit mAb designated 28-8, 28-1, 28-12, 29-8 or 20-12. Inmore preferred embodiments, the mAb is the rabbit mAb designated 28-8 oran antigen-binding portion thereof. In further embodiments, the mAb isan Ab comprising a heavy chain variable region (V_(H)) comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 35 and a light chain variable region (V_(κ)) comprisingconsecutively linked amino acids having the sequence set forth in SEQ IDNO: 36. In other embodiments, the mAb comprises the CDR1, CDR2 and CDR3domains in a V_(H) having the sequence set forth in SEQ ID NO: 35, andthe CDR1, CDR2 and CDR3 domains in a V_(κ) having the sequence set forthin SEQ ID NO: 36.

It is known in the art that rabbit Abs have certain advantages overmurine Abs. For example, rabbit Abs generally exhibit more diverseepitope recognition, improved immune response to small-size epitopes,and higher specificity and affinity compared to murine Abs (see, e.g.,Fischer et al., 2008; Cheang et al., 2006; Rossi et al., 2005). Forexample, the rabbit's lower immune dominance and larger B-cellrepertoire results in greater epitope recognition compared to murineAbs. Further, the high specificity and novel epitope recognition ofrabbit antibodies translates to success with recognition ofpost-translational modifications (Epitomics, 2013). In addition, manyprotein targets relevant to signal transduction and disease are highlyconserved between mice, rats and humans, and can therefore be recognizedas self-antigens by a mouse or rat host, making them less immunogenic.This problem is avoided by generating Abs in rabbits. Moreover, inapplications in which two antigen-specific Abs are required, it is moreconvenient to have the Abs come from two different species. Thus, forexample, it is easier to multiplex a rabbit Ab such as 28-8 with otherAbs (likely to be murine Abs since the best immune marking Abs aremurine Abs) that can mark immune cells that also express PD-L1 (e.g.,macrophages and lymphocytes). Accordingly, rabbit anti-hPD-L1 mAbs,e.g., 28-8, are particularly suited to IHC assays for detectingsurface-expressed PD-L1 in FFPE tissue samples and potentially havedistinct advantages over murine Abs, such as 5H1.

As described in Example 9, a large number (185) of Ab multiclones fromboth rabbit and mouse immunizations were screened, and only ten rabbitAb, but no mouse Ab, multiclones specifically detected the membranousform of hPD-L1. After further extensive screening by multiple rounds ofIHC, 15 purified rabbit subclones were selected based on theirspecificity and intensity of staining (see Table 5). Following furthercharacterization of the antibodies to determine their binding affinityand cross-competition by surface plasmon resonance, as well as screeningby IHC on FFPE tissues, mAb 28-8 was selected as the Ab with the bestcombination of binding to membranous PDF-L1 with high affinity andspecificity, and low background staining.

In certain aspects of this invention, the mAb or antigen-binding portioncross-competes with mouse mAb 5H1 for binding to PD-L1, which indicatesthat these antibodies bind to the same epitope region of PD-L1. Incertain other aspects, the mAb or antigen-binding portion thereof doesnot cross-compete with mouse mAb 5H1 for binding to PD-L1, indicatingthat they do not bind to the same epitope region of PD-L1.

The disclosure also provides nucleic acids encoding all of the rabbitanti-hPD-L1 Abs or portions thereof disclosed herein.

Immunotherapeutic Methods Comprising Measurement of Cell Surface PD-L1Expression

The availability of rabbit Abs that bind with high affinity specificallyto membranous PD-L1 in FFPE tissue specimens facilitates methodscomprising a step of detecting PD-L1 polypeptide on the surface of cellsin FFPE tissue samples. Accordingly, this disclosure also provides amethod for immunotherapy of a subject afflicted with cancer, whichmethod comprises: (a) selecting a subject that is a suitable candidatefor immunotherapy, the selecting comprising: (i) optionally providing aFFPE test tissue sample obtained from a patient with cancer of thetissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (ii) assessing the proportion ofcells in the test tissue sample that express PD-L1 on the cell surfaceby IHC using a rabbit anti-human PD-L1 Ab, e.g., mAb 28-8, to bind tothe PD-L1; and (iii) selecting the subject as a suitable candidate basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface exceeds a predetermined thresholdlevel; and (b) administering a composition comprising a therapeuticallyeffective amount of an agent that inhibits an inhibitoryimmunoregulator, e.g., an anti-PD-1 Ab, to the selected subject.

In certain embodiments of methods employing IHC to assay PD-L1expression in FFPE tissues, an automated IHC assay is used. Theautomated IHC process is performed on an autostainer and comprises: (a)de-paraffinizing the FFPE sample with xylene and rehydrating the sample;(b) retrieving the antigen using a decloaking chamber; (c) blockingnonspecifc protein binding sites by incubation with a Protein Block; (d)incubating the sample with a primary anti-PD-L1 Ab; (e) adding apolymeric horseradish peroxidase (HRP)-conjugated secondary Ab; (f)detecting the bound secondary Ab comprising staining with a3,3′-diaminobenzidine (DAB) chromogen; and/or (g) counterstaining withhematoxylin. This automated IHC process has been optimized byminimization of the number of steps, optimization of incubation times,and selection of primary Abs, blocking and detection reagents thatproduce strong specific staining with a low level of backgroundstaining. In preferred embodiments of this automated IHC assay, theprimary anti-PD-L1 Ab is rabbit mAb 28-8 or murine mAb 5H1. In certainembodiments of this invention, this IHC assay, and any other IHC assaydescribed herein to measure PD-L1 expression, may be used as part of amethod of immunotherapy. In other embodiments, any of the IHC methodsdescribed herein is used independently of any therapeutic processrequiring the administration of a therapeutic, i.e., solely as adiagnostic method to assay PD-L1 expression.

In certain embodiments any of the immunotherapy methods describedherein, the Ab administered to the selected subject is any anti-PD-1 oranti-PD-L1 Ab or antigen-binding portion thereof of the invention. Incertain preferred embodiments, the subject is a human. In otherpreferred embodiments, the Ab is a human Ab or antigen-binding portionthereof. In more preferred embodiments, the anti-PD-1 Ab is nivolumaband the anti-PD-L1 Ab is BMS-936559. In certain other embodiments, theanti-PD-1 Ab is an Ab or antigen-binding portion thereof thatcross-competes with nivolumab for binding to PD-1, and the anti-PD-L1 Abis an Ab or antigen-binding portion thereof that cross-competes withBMS-936559 for binding to PD-L1. In certain preferred embodiments, thecancer to be treated is selected from the group consisting MEL, RCC,squamous NSCLC, non-squamous NSCLC, CRC, castration-resistant prostatecancer CRPC, HCC, squamous cell carcinoma of the head and neck,carcinomas of the esophagus, ovary, gastrointestinal tract and breast,and a hematological malignancy.

In certain embodiments of the disclosed methods, the predeterminedthreshold is based on a proportion of (a) tumor cells, (b)tumor-infiltrating inflammatory cells, (c) particular tumor-infiltratinginflammatory cells, e.g., TILs or macrophages, or (d) a combination oftumor cells and tumor-infiltrating inflammatory cells, in a test tissuesample that expresses PD-L1 on the cell surface. In certain embodiments,the predetermined threshold is at least 0.001% of tumor cells expressingmembranous PD-L1 as determined by IHC. In other embodiments, thepredetermined threshold is at least 0.01%, preferably at least 0.1%,more preferably at least 1% of tumor cells expressing membranous PD-L1,as determined by IHC. In certain embodiments, the predeterminedthreshold is at least 5% of tumor cells expressing membranous PD-L1 asdetermined by IHC. In certain embodiments, the predetermined thresholdis at least 0.01%, at least 0.1%, at least 1%, or at least 5% of tumorcells expressing membranous PD-L1 as determined by IHC, and/or a singletumor-infiltrating inflammatory cell expressing membranous PD-L1 asdetermined by IHC. In certain other embodiments, the predeterminedthreshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5%of a tumor-infiltrating inflammatory cell expressing membranous PD-L1 asdetermined by IHC. In certain other embodiments, the predeterminedthreshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5%of a tumor-infiltrating lymphocyte expressing membranous PD-L1 asdetermined by IHC. In certain other embodiments, the predeterminedthreshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5%of a tumor-infiltrating macrophage expressing membranous PD-L1 asdetermined by IHC. In yet other embodiments, the predetermined thresholdis at least a single tumor cell or a single tumor-infiltratinginflammatory cell expressing membranous PD-L1 as determined by IHC.Preferably, PD-L1 expression is assayed by automated IHC using mAb 28-8or 5H1 as the primary Ab.

This disclosure also provides a method for treatment of a subjectafflicted with cancer, which method comprises: (a) screening a pluralityof subjects to identify a subject that is not a suitable candidate forimmunotherapy comprising the administration of an agent that inhibits aninhibitory immunoregulator, e.g., an anti-PD-1 Ab, to the subject, thescreening comprising: (i) optionally providing test tissue samples fromthe plurality of subjects, the test tissue samples comprising tumorcells and tumor-infiltrating inflammatory cells; (ii) assessing theproportion of cells in the test tissue samples that express PD-L1 on thesurface of the cells; and (iii) selecting the subject as a candidatethat is not suitable for immunotherapy based on an assessment that theproportion of cells that express PD-L1 on the surface of cells in thesubject's test tissue sample is below a predetermined threshold level;and (b) administering a standard-of-care therapeutic other than an agentthat inhibits an inhibitory immunoregulator, e.g., an anti-PD-1 Ab, tothe selected subject.

This disclosure further provides a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) screening a pluralityof subjects to identify a subject that is a suitable candidate forimmunotherapy, the screening comprising: (i) optionally providing testtissue samples from the plurality of subjects, the test tissue samplescomprising tumor cells and tumor-infiltrating inflammatory cells; (ii)assessing the proportion of cells in the test tissue samples thatexpress PD-L1 on the surface of the cells; and (iii) selecting thesubject as a candidate that is suitable for immunotherapy with an agentthat inhibits an inhibitory immunoregulator, e.g., an anti-PD-1 Ab,based on an assessment that the proportion of cells in the test tissuesample that express PD-L1 on the cell surface exceeds a predeterminedthreshold level; and (b) administering a composition comprising atherapeutically effective amount of said agent to the selected subject.

This disclosure additionally provides a method for treatment of asubject afflicted with cancer, which method comprises: (a) screening aplurality of subjects to identify a subject that is a suitable candidatefor the treatment, the screening comprising: (i) optionally providingtest tissue samples from the plurality of subjects, the test tissuesamples comprising tumor cells and tumor-infiltrating inflammatorycells; (ii) assessing the proportion of cells in the test tissue samplesthat express PD-L1 on the surface of the cells, wherein the subject isidentified as a suitable candidate for anti-PD-1 Ab immunotherapy if theproportion of cells in the tissue sample that express PD-L1 on the cellsurface exceeds a predetermined threshold level, and the subject isidentified as a candidate that is not a suitable candidate for anti-PD-1Ab immunotherapy if the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface is below a predeterminedthreshold level; and (b) administering a composition comprising atherapeutically effective amount of an agent that inhibits an inhibitoryimmunoregulator, e.g., an anti-PD-1 Ab, to the subject identified as asuitable candidate for anti-PD-1 Ab immunotherapy, or (c) administeringa standard-of-care therapeutic other than said agent to the subjectidentified as not a suitable candidate for anti-PD-1 Ab immunotherapy.

One aspect of this invention is a method for immunotherapy of a subjectafflicted with cancer, which method comprises: (a) optionally providinga test tissue sample obtained from a patient with cancer of the tissue,the test tissue sample comprising tumor cells and tumor-infiltratinginflammatory cells; (b) determining that the proportion of cells in thetest tissue sample that express PD-L1 on the cell surface is above apredetermined threshold level; and (c) based on that determinationadministering a composition comprising a therapeutically effectiveamount of an agent that inhibits an inhibitory immunoregulator, e.g., ananti-PD-1 Ab, to the subject. Another aspect of the invention is amethod for treatment of a subject afflicted with cancer, which methodcomprises: (a) optionally providing a test tissue sample obtained from apatient with cancer of the tissue, the test tissue sample comprisingtumor cells and tumor-infiltrating inflammatory cells; (b) determiningthat the proportion of cells in the test tissue sample that expressPD-L1 on the cell surface is below a predetermined threshold level; and(c) based on that determination administering a standard-of-caretherapeutic other than an agent that inhibits an inhibitoryimmunoregulator, e.g., an anti-PD-1 Ab, to the subject.

Yet another aspect of the invention is a method for immunotherapy of asubject afflicted with cancer, which method comprises: (a) optionallyproviding a test tissue sample obtained from a patient with cancer ofthe tissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (b) determining the proportion ofcells in the test tissue sample that express PD-L1 on the cell surface;(c) selecting an agent that inhibits an inhibitory immunoregulator,e.g., an anti-PD-1 Ab, as a treatment for the subject based on therecognition that an anti-PD-1 Ab is effective in patients whose testtissue sample contains a proportion of cells above a predeterminedthreshold level that express PD-L1 on the cell surface; and (d)administering a composition comprising a therapeutically effectiveamount of said agent to the subject. In another embodiment, thisdisclosure provides a method for treatment of a subject afflicted withcancer, which method comprises: (a) optionally providing a test tissuesample obtained from a patient with cancer of the tissue, the testtissue sample comprising tumor cells and tumor-infiltrating inflammatorycells; (b) determining the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface; (c) selecting a standard-of-caretherapeutic other than an agent that inhibits an inhibitoryimmunoregulator, e.g., an anti-PD-1 Ab, as a treatment for the subjectbased on the recognition that said agent is ineffective in patients inwhom the proportion of cells that express PD-L1 on the cell surface inthe test tissue sample is below a predetermined threshold level; and (d)administering the standard-of-care therapeutic to the subject.

This disclosure also provides a method of selecting an immunotherapy fora subject afflicted with cancer, which method comprises: (a) assayingcells of a test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells to assess the proportion of cellsin the test tissue sample that express PD-L1; and (b) based on anassessment that the proportion of cells that express membranous PD-L1 isabove a predetermined threshold level, selecting an immunotherapycomprising a therapeutically effective amount of an agent that inhibitsan inhibitory immunoregulator, e.g., an anti-PD-1 Ab, for the subject.The disclosure further provides a method of selecting a treatment for asubject afflicted with cancer, which method comprises: (a) assayingcells of a test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells to assess the proportion of cellsin the test tissue sample that express PD-L1; and (b) based on anassessment that the proportion of cells that express membranous PD-L1 isbelow a predetermined threshold level, selecting a standard-of-caretreatment other than an agent that inhibits an inhibitoryimmunoregulator, e.g., an anti-PD-1 Ab, for the subject.

In addition, this disclosure provides a method for treatment of asubject afflicted with cancer, which method comprises administering tothe subject a composition comprising a therapeutically effective amountof an agent that inhibits an inhibitory immunoregulator, e.g., ananti-PD-1 Ab, the subject having been selected on the basis that theproportion of cells in a test tissue sample from the subject thatexpress PD-L1 is determined to exceed a predetermined threshold level,wherein the test tissue sample comprises tumor cells andtumor-infiltrating inflammatory cells. This disclosure also provides amethod for treatment of a subject afflicted with cancer, which methodcomprises administering to the subject a standard-of-care treatmentother than an agent that inhibits an inhibitory immunoregulator, e.g.,an anti-PD-1 Ab, the subject having been selected on the basis that theproportion of cells in a test tissue sample from the subject thatexpress PD-L1 is determined to be below a predetermined threshold level,wherein the test tissue sample comprises tumor cells andtumor-infiltrating inflammatory cells.

This disclosure further provides a method for selecting a cancer patientfor immunotherapy with an agent that inhibits an inhibitoryimmunoregulator, e.g., an anti-PD-1 Ab, which method comprises: (a)optionally providing a test tissue sample obtained from a patient withcancer of the tissue, the test tissue sample comprising tumor cells andtumor-infiltrating inflammatory cells; (b) assaying the test tissuesample to determine the proportion of cells therein that express PD-L1on the cell surface; (c) comparing the proportion of cells that expressPD-L1 on the cell surface with a predetermined threshold proportion; and(d) selecting the patient for immunotherapy based on an assessment thatthe proportion of cells in the test tissue sample that express surfacePD-L1 is above the predetermined threshold level.

In any of the methods described herein comprising a step for assessingPD-L1 expression, the test tissue sample may be a FFPE tissue sample andPD-L1 polypeptide on the cell surface is detected by IHC using ananti-PD-L1 Ab, e.g., mAb 28-8 or 5H1.

In addition, in any method where an immunotherapy is selected oradministered based on an assessment that the proportion of cells in atest tissue sample from the subject expresses PD-L1 at a level above apredetermined threshold level, it follows that a complementary method oftreatment may be performed wherein a standard-of-care treatment otherthan the immunotherapy is selected or administered based on anassessment that the proportion of cells in a test tissue sample from thesubject expresses PD-L1 at a level below the predetermined thresholdlevel.

This disclosure further provides a method for predicting the therapeuticeffectiveness of an agent that inhibits an inhibitory immunoregulator,e.g., an anti-PD-1 Ab, for treating a cancer patient, which methodcomprises: (a) optionally providing a test tissue sample obtained from apatient with cancer of the tissue, the test tissue sample comprisingtumor cells and tumor-infiltrating inflammatory cells; (b) assaying thetest tissue sample to determine the proportion of cells therein thatexpress PD-L1 on the cell surface; (c) comparing the proportion of cellsthat express PD-L1 on the cell surface with a predetermined thresholdvalue; and (d) predicting the therapeutic effectiveness of said agent,wherein if the proportion of cells that express PD-L1 on the cellsurface exceeds the threshold proportion the agent is predicted to beeffective in treating the patient, and wherein if the proportion ofcells that express PD-L1 on the cell surface is below the thresholdproportion the agent is predicted to not be effective in treating thepatient.

This disclosure also provides a method for determining animmunotherapeutic regimen comprising an agent that inhibits aninhibitory immunoregulator, e.g., an anti-PD-1 Ab, for treating a cancerpatient, which method comprises: (a) optionally providing a test tissuesample obtained from a patient with cancer of the tissue, the testtissue sample comprising tumor cells and tumor-infiltrating inflammatorycells; (b) assaying the test tissue sample to determine the proportionof cells therein that express PD-L1 on the cell surface; (c) comparingthe proportion of cells that express PD-L1 on the cell surface with apredetermined threshold proportion; and (d) determining animmunotherapeutic regimen comprising said agent based on thedetermination that the proportion of cells that express PD-L1 on thecell surface exceeds the predetermined threshold proportion.

Standard-of-Care Therapeutics

Several of the methods of treatment described herein comprise theadministration of a standard-of-care therapeutic to a patient. As usedherein, a “standard-of-care therapeutic” is a treatment process,including a drug or combination of drugs, radiation therapy (RT),surgery or other medical intervention that is recognized by medicalpractitioners as appropriate, accepted, and/or widely used for a certaintype of patient, disease or clinical circumstance. Standard-of-caretherapies for different types of cancer are well known by persons ofskill in the art. For example, the National Comprehensive Cancer Network(NCCN), an alliance of 21 major cancer centers in the USA, publishes theNCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES®) thatprovide detailed up-to-date information on the standard-of-caretreatments for a wide variety of cancers (see NCCN GUIDELINES®, 2013).By way of example, standard-of-care treatments for MEL, RCC and NSCLCare summarized below.

Melanoma

MEL is a malignant tumor of melanocytes, the melanin-producing cellsfound predominantly in skin. Though less common than other skin cancers,it is the most dangerous of skin cancers if not diagnosed early andcauses the majority (75%) of skin cancer deaths. The incidence of MEL isincreasing worldwide in Caucasian populations, especially where peopleswith low amounts of skin pigmentation receive excessive ultravioletlight exposure from the sun. In Europe, the incidence rate is <10-20 per100,000 population; in the USA 20-30 per 100,000; and in Australia,where the highest incidence is observed, 50-60 per 100,000 (Garbe etal., 2012). MEL accounts for about 5% of all new cases of cancer in theUnited States (U.S.), and the incidence continues to rise by almost 3%per year. This translates to an estimated 76,690 new cases in the U.S.in 2013 with 9,480 associated deaths (Siegel et al. (2013).

For in situ (stage 0) or early-stage MEL (Stages I-II), surgicalexcision is the primary treatment. In general, the prognosis isexcellent for patients with localized disease and tumors 1.0 mm or lessin thickness, with 5-year survival rates of more than 90% (NCCNGUIDELINES®, 2013—Melanoma). Where surgical excision is not feasible forin situ melanoma due to comorbidity or cosmetically sensitive tumorlocation, topical imiquimod and radiotherapy are emerging as treatments,especially for lentigo maligna. Chemotherapeutic agents for treating MELinclude dacarbazine, temozolomide and imatinib for melanoma with a c-KITmutation, high-dose interleukin-2, and paclitaxel with or withoutcarboplatin. However, these treatments have modest success, withresponse rates below 20% in first-line (1L) and second-line (2L)settings.

For patients with localized melanomas more than 1.0 mm in thickness,survival rates range from 50-90%. The likelihood of regional nodalinvolvement increases with increasing tumor thickness. With Stage IIIMEL (clinically positive nodes and/or in-transit disease), 5-yearsurvival rates range from 20-70%. By far the most lethal is Stage IV MELwhere long-term survival in patients with distant metastatic melanoma isless than 10% (NCCN GUIDELINES®, 2013—Melanoma).

There is no consensus on the best treatments for metastatic MEL, thougha variety of treatments including excision to clear margins,intralesional injections, laser ablation, radiation and biochemotherapy(combination of chemotherapy and biological agents such asinterferon-alpha and IL-2) are being investigated. The therapeuticlandscape for metastatic MEL has recently seen dramatic improvementswith the development of novel drugs such as vemurafenib and ipilimumab.Vemurafenib specifically inhibits signaling by a mutated intracellularkinase, BRAF, that is present in about 50% of patients with metastaticMEL. In clinical trials, vemurafenib delivered an estimatedprogression-free survival (PFS) of 5.3 months in BRAF mutation-positivepatients versus merely 1.6 months for dacarbazine, and a median OS of15.9 months for vemurafenib versus 5.6-7.8 months for dacarbazine(Chapman et al., 2011; Sosman et al., 2012). Ipilimumab is a HuMAb thatinhibits the immune checkpoint receptor, CTLA-4, and thereby stimulatesa T cell immune response. Ipilimumab, with or without a glycoprotein 100(gp100) peptide vaccine, improved median OS in patients with previouslytreated metastatic MEL to 10.0-10.1 months as compared with 6.4 monthsamong patients receiving gp100 alone (Hodi et al., 2010). Besides thesetwo agents, no other agent has demonstrated an OS benefit in a Phase 3randomized study. Dacarbazine is approved by the FDA and the EMA fortreatment of metastatic MEL with a reported objective response rate of5-20% and a median OS of about 6.4 months, but these responses areshort-lived. Other drugs such as temozolomide and fotemustine have notresulted in significant improvement in survival when compared todacarbazine. IL-2 has also been approved by the FDA for the treatment ofmetastatic MEL as it is associated with a 15-20% response rate including4-6% complete responses which can be durable, but it is associated withsignificant toxicities including hypotension, cardiac arrhythmias, andpulmonary edema. Further details on standard-of-care treatments formelanoma are provided by Garbe et al. (2012) and the NCCN GUIDELINES®,2013—Melanoma. Despite the recent approval of ipilimumab and vemurafenibfor advanced MEL, there is still a large unmet need for patients whohave progressed on anti-CTLA-4 therapy and a BRAF inhibitor (dependingon BRAF status) or patients with previously untreated, unresectable ormetastatic BRAF wild-type MEL. The 5-year survival rate for late-stageMEL is currently only 15%.

Renal Cell Carcinoma

RCC is the most common type of kidney cancer in adults, responsible forapproximately 90% of renal tumors, and 80-90% of these are clear celltumors (NCCN GUIDELINES®, 2013—Kidney Cancer). It is also the mostlethal of all the genitourinary tumors. An estimated 65,150 patientswill be diagnosed with renal cancer and 13,680 will die of the diseasein the United States in 2013 (Siegel et al. (2013).

For clinically localized RCC (Stage IA and IB), surgical resection,including radical nephrectomy and nephron-sparing surgery, is aneffective therapy. Partial nephrectomy is generally not suitable forpatients with locally advanced tumors (Stage II and III), in which caseradical nephrectomy is preferred. Where the tumor is confined to therenal parenchyma, the 5-year survival rate is 60-70%, but this islowered considerably in Stage IV disease where metastases have spread.Stage IV RCC is relatively resistant to RT and chemotherapy, althoughpatients may benefit from surgery, and cytoreductive nephrectomy beforesystemic therapy is recommended for patients with a potentiallysurgically resectable primary and multiple resectable metastases.

Until recently, the cytokines IL-2 and IFNα were the only activesystemic treatments for advanced or metastatic RCC, both providingreported ORRs of 5-27%. However, due to each of these agent's limitedclinical benefit and substantial toxicity profile, newer targeted agentshave largely replaced cytokines in the treatment of advanced ormetastatic renal cell carcinoma. The recognition of the importance ofhypoxia inducible factor alpha (HIFα) signaling in the pathogenesis ofclear-cell RCC has led to widespread study of two classes of targetedtherapies, anti-angiogenic tyrosine kinase inhibitors (TKIs) andmammalian target of rapamycin (mTOR) inhibitors, in 1L and 2L treatments(Mulders, 2009). Targeting of angiogenesis is rational becauseconstitutive HIFα activation leads to the upregulation or activation ofseveral proteins including vascular endothelial growth factor (VEGF),which can subsequently lead to tumor proliferation and neovasculatureformation. Targeting of the mTOR pathway is important because activationof the upstream PI3K/Akt/mTOR signaling pathway is one method by whichconstitutive HIFα activation or upregulation occurs (Mulders, 2009).Agents that target angiogenesis include VEGF-receptor (VEGFr) TKIs(e.g., sorafenib, sunitinib, pazopanib, axitinib, and tivozanib) andVEGF-binding mAbs (e.g., bevacizumab), while agents that target the mTORpathway include the mTOR inhibitors (e.g., everolimus and temsirolimus)(Mulders, 2009; NCCN GUIDELINES®, 2013—Kidney Cancer). However, mostpatients develop resistance, and OS improvement has only been shown inone phase 3 trial in poor-risk patients: temsirolimus showed astatistically significant benefit for OS in patients with advanced RCCcompared to IFNα (10.9 months versus 7.3 months) (Hudes et al., 2007).Everolimus has also demonstrated a 2.1-month improvement in median PFSversus placebo, but with no OS improvement (Motzer et al., 2008). Amongthe five approved anti-angiogenic agents (sorafenib, sunitinib,bevacizumab, pazopanib, and axitinib) and two approved mTOR inhibitors(temsirolimus, everolimus), only everolimus is approved specifically foruse after the failure of treatment with anti-angiogenic therapy. In theU.S., everolimus is indicated for the treatment of advanced RCC afterfailure of treatment with sunitinib or sorafenib, whereas in the EU,everolimus is more broadly indicated for patients with advanced RCC,whose disease has progressed on or after treatment with VEGF-targetedtherapy.

Non-Small Cell Lung Cancer

NSCLC is the leading cause of cancer death in the U.S. and worldwide,exceeding breast, colon and prostate cancer combined. In the U.S., anestimated 228,190 new cases of lung and bronchial will be diagnosed inthe U.S., and some 159,480 deaths will occur because of the disease(Siegel et al., 2013). The majority of patients (approximately 78%) arediagnosed with advanced/recurrent or metastatic disease. Metastases tothe adrenal gland from lung cancer are a common occurrence, with about33% of patients having such metastases. NSCLC therapies haveincrementally improved OS, but benefit has reached a plateau (median OSfor late stage patients is just 1 year). Progression after 1L therapyoccurred in nearly all of these subjects and the 5-year survival rate isonly 3.6% in the refractory setting. From 2005 to 2009, the overall5-year relative survival rate for lung cancer in the U.S. was 15.9%(NCCN GUIDELINES®, 2013—Non-Small Cell Lung Cancer).

Surgery, RT and chemotherapy are the three modalities commonly used totreat NSCLC patients. As a class, NSCLCs are relatively insensitive tochemotherapy and RT, compared to small cell carcinoma. In general, forpatients with Stage I or II disease, surgical resection provides thebest chance for cure, with chemotherapy increasingly being used bothpre-operatively and post-operatively. RT can also be used as adjuvanttherapy for patients with resectable NSCLC, the primary local treatment,or as palliative therapy for patients with incurable NSCLC.

Patients with Stage IV disease who have a good performance status (PS)benefit from chemotherapy. Many drugs, including platinum agents (e.g.,cisplatin, carboplatin), taxanes agents (e.g., paclitaxel, albumin-boundpaclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexedand gemcitabine are useful for Stage IV NSCLC. Combinations using manyof these drugs produce 1-year survival rates of 30% to 40% and aresuperior to single agents. Specific targeted therapies have also beendeveloped for the treatment of advanced lung cancer. For example,bevacizumab (AVASTIN®) is a mAb that blocks vascular endothelial growthfactor A (VEGF-A). Erlotinib (TARCEVA®) is a small-molecule TKI ofepidermal growth factor receptor (EGFR). Crizotinib (XALKORI®) is asmall-molecule TKI that targets ALK and MET, and is used to treat NSCLCin patients carrying the mutated ALK fusion gene. Cetuximab (ERBITUX®)is a mAb that targets EGFR.

There is a particular unmet need among patients who have squamous cellNSCLC (representing up to 25% of all NSCLC) as there are few treatmentoptions after 1L therapy. Single-agent chemotherapy is standard of carefollowing progression with platinum-based doublet chemotherapy(Pt-doublet), resulting in median OS of approximately 7 months.Docetaxel remains the benchmark treatment in this line of therapyalthough erlotinib may also be used with less frequency. Pemetrexed hasalso been shown to produce clinically equivalent efficacy outcomes butwith significantly fewer side effects compared with docetaxel in the 2Ltreatment of patients with advanced NSCLC (Hanna et al., 2004). Notherapy is currently approved for use in lung cancer beyond the 3Lsetting. Pemetrexed and bevacizumab are not approved in squamous NSCLC,and molecularly targeted therapies have limited application. The unmetneed in advanced lung cancer has been compounded by the recent failureof Oncothyreon and Merck KgaA's STIMUVAX® to improve OS in a phase 3trial, inability of ArQule's and Daiichi Sankyo's c-Met kinaseinhibitor, tivantinib, to meet survival endpoints, failure of EliLilly's ALIMTA® in combination with Roche's AVASTIN® to improve OS in alate-stage study, and Amgen's and Takeda Pharmaceutical's failure tomeet clinical endpoints with the small-molecule VEGF-R antagonist,motesanib, in late-stage trials.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allreferences cited throughout this application are expressly incorporatedherein by reference.

Example 1 Cross-Competition Between Anti-PD-1 HuMAbs for Binding to CHOCells Expressing Human PD-1

Chinese Hamster Ovary (CHO) cells transfected to express human PD-1(CHO/PD-1 cells) were incubated with 10 μg/ml of Fab fragment of theanti-PD-1 HuMAb 5C4 or human IgG1 (hIgG1) isotype control Ab for 30minutes at 4° C. before addition of anti-PD-1 HuMAbs 2D3, 7D3 or 4H1 ata concentration of 0.2 μg/ml. Binding of 4H1, 2D3 or 7D3 to CHO/PD-1cells were detected by fluorescein isothiocyanate (FITC)-conjugated goatanti-hIgG, Fc-gamma specific Ab. In the case of cross-competition assaywith 5C4 and 17D8, CHO/PD-1 cells were incubated with the whole moleculeof 5C4 before addition of FITC-labeled 17D8. Binding of 2D3, 7D3, 4H1 or17D8 to the CHO/PD-1 cells was measured by flow cytometric analysisusing a FACScalibur flow cytometer (Becton Dickinson, San Jose, Calif.).

The results are depicted in FIG. 1. The data show that the 5C4 Fabfragment substantially blocked the binding of mAbs 5C4 itself, as wellas the binding of 2D3, 7D3 (FIG. 1A) and 4H1 (FIG. 1B), while the 5C4whole mAb substantially blocked the binding of 17D8 (FIG. 1C) toCHO/PD-1 cells as measured by mean fluorescence intensity (MFI) ofstaining.

Example 2 Cross-Competition Between Anti-PD-L1 HuMAbs for Binding to CHOCells Expressing Human PD-L1

CHO cells transfected to express hPD-L1 (CHO/PD-L1 cells) were incubatedwith 10 μg/ml of each of ten unconjugated human anti-PD-L1 mAbs (5F8,7H1, 10H10, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7, and 13G4) or human IgG1(hIgG1) isotype control Ab for 20 min at 4° C. FITC-conjugated 10H10(A), 3G10 (B), 10A5 (C), 11E6 (D), 12A4 (E) or 13G4 (F) was added to thecells to a final concentration of 0.09 μg/ml (B, D), 0.27 μg/ml (A, C),0.91 μg/ml (F), or 2.73 μg/ml (E) for an additional 20 min at 4° C.without prior washout of unbound, unconjugated Ab. Different quantitiesof the various FITC-conjugated HuMAbs were used due to differences inbinding efficiency following labeling, and the optimal amounts of theseFITC-conjugated HuMAbs were previously determined by dose-titrationanalysis of binding to CHO/PD-L1 cells. Binding of FITC-conjugated10H10, 3G10, 10A5, 11E6, 12A4 or 13G4 to the CHO/PD-L1 cells wasmeasured by flow cytometry.

The results are depicted in FIG. 2. Binding of labeled 10H10 waspartially blocked by 10A5, 11E6 and 13G4, but was substantially blockedonly by itself (FIG. 2A). Conversely, 10H10 substantially blocked thebinding only of itself to CHO/PD-L1 cells. Each of anti-PD-L1 HuMAbs5F8, 7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7 and 13G4 substantiallyblocked binding of labeled mAbs 3G10 (FIG. 2B), 10A5 (FIG. 2C), 11E6(FIG. 2D), 12A4 (FIG. 2E) and 13G4 (FIG. 2F) to CHO/PD-L1 cells asmeasured by MFI, though mAbs 5F8 and 13G4 generally blocked binding ofthe labeled mAbs to a slightly lesser extent.

Example 3 Cross-Competition Between Anti-PD-L1 mAbs for Binding toOvarian Carcinoma Cells Expressing Human PD-L1

Anti-PD-L1 HuMAbs 5F8, 12B7, 3G10, 1B12, 13G4, 10H10, 10A5 and 12A4, anda human IgG1 (huIgG1) isotype control Ab were serially diluted from 10μg/ml and incubated with hPD-L1-expressing ES-2 ovarian carcinoma cellsfor 20 minutes at 4° C. Without washing, biotinylated-12A4 Ab was addedto a final concentration of 0.4 μg/ml for an additional 20 minutes at 4°C. After washing, bound biotin-12A4 was detected using fluorescentstreptavidin-PE secondary reagent and measured by flow cytometry. FIG. 3shows the fluorescence of bound biotin-12A4 plotted against theconcentration of unlabeled hPD-L1 HuMAbs. Binding of biotin-12A4 to ES-2cells was substantially blocked by 12A4 itself and by 1B12 and 12B7, andwas moderately to significantly blocked by mAbs 5F8, 10A5, 13G4 and3G10, but was not blocked by mAb 10H10.

Example 4 Design of Phase 1 Clinical Study of Anti-PD-1 Ab

A Phase 1 study was conducted to assess the safety, antitumor activity,and pharmacokinetics of an anti-PD-1 in patients with selected advancedsolid tumors. The human anti-PD-1 mAb, BMS-936558 (also referred toherein as nivolumab, and in U.S. Pat. No. 8,008,449 as 5C4), wasadministered as an intravenous infusion every 2 weeks of each 8-weektreatment cycle. Tumor status was re-assessed following each cycle.Patients continued treatment for up to 2 years (12 cycles), until theyexperienced complete remission, unacceptable toxicity, diseaseprogression, or withdrew consent. In patients who were otherwiseclinically stable, study treatment was continued beyond apparent initialdisease progression until further progression was noted as recommendedby proposed immune response criteria (Wolchok et al., 2009). Patientswith stable disease (SD) or an ongoing objective response (OR: completeresponse [CR] or partial response [PR]) at the end of treatment werefollowed for 1 year and were offered retreatment for 1 additional yearin the event of progression.

Dose Escalation

Patients with advanced melanoma (MEL), non-small cell lung cancer(NSCLC), renal cell carcinoma (RCC), castration-resistant prostatecancer (CRPC) and colorectal cancer (CRC) were eligible to enroll.Cohorts of 3-6 patients per dose level were enrolled sequentially at1.0, 3.0, and 10.0 mg/kg. Dose escalation proceeded when a minimum of 3patients had completed the safety evaluation period (56 days) at thegiven dose level, with dose-limiting toxicity in less than one-third ofpatients. Intra-patient dose escalation was not permitted.

Cohort Expansion

A maximum tolerated dose (MTD) was not reached. Initially, 5 expansioncohorts of approximately 16 patients each were enrolled at 10 mg/kg forMEL, NSCLC, RCC, CRPC and CRC. Based on initial signals of activity, andfollowing a 6.5-month hiatus for protocol amendment, additionalexpansion cohorts of approximately 16 patients each were enrolled forMEL (at 1.0 and 3.0 mg/kg, followed by cohorts randomized to 0.1, 0.3,or 1.0 mg/kg), NSCLC (squamous or nonsquamous histology cohortsrandomized to 1, 3, or 10 mg/kg), and RCC (at 1.0 mg/kg). Intra-patientdose escalation to 1 mg/kg was permitted for MEL patients withprogressive disease after receiving 0.1 or 0.3 mg/kg.

Patients

Eligible patients had documented advanced solid tumors; age >18 years;life expectancy >12 weeks; Eastern Cooperative Oncology Groupperformance status of ≦2; measurable disease by Response EvaluationCriteria in Solid Tumors (RECIST), v1.0 with modification (see Topalianet al., 2012b); adequate hematologic, hepatic, and renal function; andreceived 1-5 prior systemic treatment regimens. Patients with stabletreated brain metastases were enrolled. Exclusion criteria included ahistory of chronic autoimmune disease, prior therapy with T-cellmodulating Abs (e.g., anti-CTLA-4, anti-PD-1, anti-PD-L1), conditionsrequiring immunosuppressive medications, and chronic infections (e.g.,HIV, hepatitis B or C).

A total of 296 patients with advanced solid tumors including MEL(n=104), NSCLC (n=122), RCC (n=34), CRPC (n=17), and CRC (n=19) weretreated with BMS-936558 for 40 months up to February 2012. By March2013, 306 patients including patients with non-small cell lung cancer(n=129), melanoma (n=107), RCC (n=34), CRPC (n=17), and CRC (n=19) hadbeen treated with BMS-936558 from October 2008 through January 2012, allwith a minimum of one year of observation. Two patients did not receivea full cycle of treatment and were not considered response-evaluable.Median age was 63 years (range, 29-85). ECOG performance score was 0 or1 in 98% of patients. The majority of patients were heavily pretreated,with 47% having received at least 3 prior regimens. Notable priortherapies included immunotherapy (64%) and B-RAF inhibitor (8%) in MELpatients; platinum-based chemotherapy (94%) and tyrosine kinaseinhibitors (TKIs, 34%) in NSCLC patients; and nephrectomy (94%),immunotherapy (59%), and anti-angiogenic therapy (74%) in RCC patients.Baseline characteristics of the total treated population (N=306) weresimilar to those of the efficacy population (response evaluablepatients, N=270). Details on the patient pre-treatments are provided inTopalian et al. (2012b).

Statistical Analysis

Baseline characteristics and adverse events for all treated patients(N=306), and efficacy results for 270 patients with lung cancer,melanoma, and kidney cancer as of March 2013 are reported.Pharmacokinetic and molecular-marker populations consisted of treatedpatients with available data as of the February 2012 date of preliminaryanalysis. The efficacy population consisted of response-evaluablepatients commencing treatment at least 8 months before the date ofanalysis. Tumor measurements were collected after each treatment cycle(4 doses) by investigators. Individual best objective responses based onthe tumor measurements were assessed by the sponsor per modified RECISTv1.0. Objective response was confirmed by at least one sequential tumorassessment. Objective response and stable disease rates were estimatedwith confidence intervals using the Clopper-Pearson method.Time-to-event endpoints including PFS, OS, survival rates, and durationof response, were estimated using the Kaplan-Meier method. AEs werecoded using Medical Dictionary for Regulatory Activities (MedDRA),version 15.1. Select adverse events with potential immunologicetiologies, also referred to as “immune-related adverse events” of “AEsof special interest” (AEOSIs), defined as adverse events that requiremore frequent monitoring and/or unique intervention, were identifiedusing a pre-defined list of MeDRA terms. Individual best ORs werederived from investigator-reported data per modified RECIST v1.0. OR wasconfirmed by at least one sequential tumor assessment and OR rate(ORR={[CR+PR]÷n}×100) was calculated.

Example 5 Safety Evaluations on Patients Treated with Anti-PD-1 Antibody

Safety evaluations, including clinical examination and laboratoryassessments, were conducted in all treated patients at baseline andregular intervals up to 100 days following last administration of drug.The severity of AEs was graded based on the NCI Common TerminologyCriteria for Adverse Events (NCI CTCAE), v3.0. Computed tomography (CT)or magnetic resonance imaging was performed for tumor assessment atbaseline and following each treatment cycle.

A MTD was not defined across the doses of BMS-936558 tested on thisstudy, up to the highest planned dose of 10 mg/kg. A relative BMS-936558dose intensity of 90% or higher was achieved in 87% of patients (seeTopalian et al., 2012b; Topalian et al., 2013, for details). AEs werecoded using Medical Dictionary for Regulatory Activities (MedDRA),version 14.1. AEOSIs were identified using a pre-defined list of MeDRAterms. Fifteen of 296 (5%) patients discontinued treatment due toBMS-936558-related AEs. As of the February 2012 date of preliminaryanalysis, 62 (21%) patients had died, and by March 2013, 195 patients(64%) had died, with disease progression being the most common cause ofdeath (Topalian et al., 2012b; Topalian et al., 2013).

The most common adverse events, regardless of causality includedfatigue, decreased appetite, diarrhea, nausea, cough, dyspnea,constipation, vomiting, rash, pyrexia and pruritus (Topalian et al.,2012b; Topalian et al., 2013). Common BMS-936558-related AEs includedfatigue, rash, diarrhea, decreased appetite, and nausea. The majority ofthe events were low grade, with grade 3-4 drug-related AEs observed in41 of 296 (14%) patients. Treatment-related AEs (any grade) wereobserved in 230 of 306 patients (75%), the most common being fatigue,rash, diarrhea and pruritus (Topalian et al., 2013). The spectrum,frequency, and severity of BMS-936558-related AEs were generally similaracross the dose levels tested, with no clear relationship to treatmentduration. Fifty-two of 306 patients (17%) experienced Grade 3-4treatment-related adverse events, with fatigue (2%), pneumonitis,lymphopenia, diarrhea, abdominal pain, and hypophosphatemia (1% each)being the most common. Treatment-related serious adverse events occurredin 42 of 306 patients (14%) (Topalian et al., 2013).

Drug-related AEOSIs, with potential immune-related etiologies, werecategorized by organ involvement to provide a more accurate estimate offrequency and included pneumonitis, vitiligo, colitis, hepatitis,hypophysitis, and thyroiditis among others. Treatment-related selectadverse events of any grade were observed in 140 of 306 patients (46%),the most common being rash (15%), diarrhea (13%), and pruritus (11%)(Table 2). Grade 3-4 treatment-related select events were seen in 19patients (6%).

Fifty-two of 230 patients (23%) with drug-related adverse eventsrequired management with systemic glucocorticoids and/or otherimmunosuppressive agents. Hepatic or gastrointestinal AEOSIs weremanaged with treatment interruption and, as necessary, withadministration of corticosteroids. Among patients treated to-date, theseAEs were reversible in all cases. Endocrine AEOSIs were managed withreplacement therapy. Thirty-two of 306 patients (11%) discontinuedtherapy due to treatment-related adverse events. At the discretion ofthe treating physician, patients successfully reinitiated treatment withBMS-936558. Twenty-one (40%) were able to resume nivolumab therapy aftertoxicity resolved.

Drug-related pneumonitis (any grade) occurred in 12 of 306 (4%) patients(Topalian et al., 2013). Clinical presentations ranged from radiographicabnormalities in asymptomatic patients, to progressive, diffusepulmonary infiltrates associated with symptoms (cough, fever, dyspnea).Grade 3-4 pneumonitis developed in 4 patients (1%), of which 3 caseswere fatal (2 NSCLC patients, 1 CRC). No clear relationship between theoccurrence of pneumonitis and tumor type, dose level, or the number ofdoses received was noted. In 9 of 12 patients, pneumonitis wasreversible with treatment discontinuation and/or immunosuppression(glucocorticoids, infliximab, mycophenolate). Following the most recentpneumonitis-associated death in November 2011, 79 patients continued toreceive nivolumab (median 29 weeks, range 2-69 weeks) with no furthermortalities from this or other treatment-related causes.

Example 6 Pharmacokinetics/Pharmacodynamics Analyses on Anti-PD-1Antibody

For pharmacokinetic (PK) analyses, serial blood samples were collectedand serum concentrations of BMS-936558 were quantified using by ELISA.For pharmacodynamic (PD) analysis, peripheral blood mononuclear cellswere isolated from patients at baseline and following cycle 1 toestimate PD-1 receptor occupancy (RO) by BMS-936558 on circulating CD3+T-cells via flow cytometry (Brahmer et al., 2010).

The maximum concentration of BMS-936558 was observed at a median T_(max)of 1-4 hours after the start of infusion. The PK of BMS-936558 waslinear with a dose proportional increase in C_(max) and AUC_(0-14 d)) inthe dose range of 0.1-10 mg/kg (n=35). BMS-936558 PD was assessed byPD-1 RO on circulating T-cells. PBMCs from 65 MEL patients treated withone cycle of BMS-936558 at 0.1-10 mg/kg biweekly demonstrated medianoccupancy of PD-1 molecules on circulating CD3⁺ T-cells by BMS-936558ranging from 64%-70% (see Topalian et al., 2012b, for details).

Example 7 Antitumor Efficacy Exhibited by Anti-PD-1 Antibody

Data Analyzed as of February 2012

Clinical antitumor activity was observed at all BMS-936558 doses tested.ORs (confirmed CR or PR) were observed in a substantial portion ofpatients with NSCLC, MEL, and RCC (Tables 1 and 2; FIG. 4), and invarious sites of metastatic disease including liver, lung, lymph nodes,and bone (FIGS. 5-7 and not shown). Tumor regressions followedconventional as well as “immune-related” patterns of response, such asprolonged reduction in tumor burden in the presence of new lesions.Individual best overall responses were derived frominvestigator-reported data according to modified RECIST v1.0. OR wasconfirmed by at least one sequential tumor assessment. At the time ofdata analysis, 2 patients with NSCLC who were treated with 10 mg/kg hadunconfirmed responses, and 8 additional patients (with MEL, NSCLC, orRCC) had a persistent reduction in baseline target lesions in thepresence of new lesions, (i.e., an “immune-related” response pattern).None of these patients was categorized as a responder for the purpose ofcalculating OR rates. Antitumor responses and/or prolonged diseasestabilization were observed in patients irrespective of prior therapiesreceived (see summary of progression free interval for patients with ORand SD in Supplementary Appendix 4 of Topalian et al., 2012b).

In NSCLC patients, 14 ORs were observed at BMS-936558 doses of 1, 3, or10 mg/kg with response rates of 6%, 32%, and 18%, respectively. ORs wereobserved across NSCLC histologies: 6 responders of 18 squamous (33%), 7responders of 56 nonsquamous (13%), and 1 of 2 unknown. All 14 patientswith ORs started treatment ≧24 weeks before data analysis, and of these,8 had response duration ≧24 weeks (Table 1). Stable disease (SD) lasting≧24 weeks was observed in 5 (7%) NSCLC patients, all with nonsquamoushistology. Among MEL patients, 26 ORs were observed at doses rangingfrom 0.1-10 mg/kg, with response rates ranging from 19%-41% per doselevel. At the 3 mg/kg dose level, ORs were noted in 7 of 17 (41%)patients. Of 26 MEL patients who achieved an OR, 17 started treatment ≧1year before data analysis, and of these, 13 patients had an OR duration≧1 yr. The remaining 8 patients with OR were on study <1 year and 6 hadresponses ranging from 1.9-5.6 months. SD lasting ≧24 weeks was observedin 6 (6%) patients. In RCC patients, ORs occurred in 4 of 17 (24%)patients treated with a BMS-936558 dose of 1 mg/kg and 5 of 16 (31%)patients treated with 10 mg/kg. Among 8 RCC patients with OR who startedtreatment ≧1 year prior to data analysis, 5 (63%) had OR duration ≧1 yr.SD lasting ≧24 weeks was observed in an additional 9 (27%) patients.

TABLE 1 Clinical Activity of BMS-936558 in the Efficacy Population* (N =236)^(†) as Assessed to February 2012 ORR^(‡) SD ≧ 24 wk PFSR^(§) DoseNo. Patients (%) No. Patients (%) at 24 wk (%) Tumor Type (mg/kg) n [95%CI]^(†) [95% CI] [95% CI] MEL 0.1 14 4 (29) [8-58] 1 (7) [0.2-34] 40[13-66] 1.0 27 8 (30) [14-50] 3 (11) [2-29] 45 [26-65] 3.0 17 7 (41)[18-67] 1 (6) [0.1-29] 55 [30-80] 10.0 20 4 (20) [6-44] 0 30 [9-51] ALLMEL 94 26 (28) [19-38] 6 (6) [2-13] 41 [30-51] NSCLC** All 1 18 1 (6)[0.1-27] 1 (6) [0.1-27] 16 [0-34] Squamous 1 5 0 0 0 Nonsquamous 1 12 01 (8) [0.2-39] 14 [0-37] Unknown 1 1 1(100) [3-100] 0 1 All 3 19 6 (32)[13-57] 2 (11) [1-33] 41 [18-64] Squamous 3 6 3 (50) [12-88] 0 50[10-90] Nonsquamous 3 13 3 (23) [5-54] 2 (15) [2-45] 37 [10-64] All 1039 7 (18) [8-34] 2 (5) [0.6-17] 24 [11-38] Squamous 10 7 3 (43) [10-82]0 43 [6-80] Nonsquamous 10 31 4 (13) [4-30] 2 (7) [0.8-21] 21 [6-36]Unknown 10 1 0 0 0 ALL NSCLC 76 14 (18) [11-29] 5 (7) [2-15] 26 [16-36]All Squamous 18 6 (33) [13-59] 0 33 [12-55] All 56 7 (13) [5-24] 5 (9)[3-20] 22 [ 11-34] Nonsquamous RCC 1 17 4 (24) [7-50] 4 (24) [7-50] 47[23-71] 10 16 5 (31) [11-59]^(‡) 5 (31) [11-59]^(¶) 67 [43-91] ALL RCC33 9 (27) [13-46] 9 (27) [13-46] 56 [39-73 *The efficacy populationconsists of response-evaluable patients whose treatment was initiated atleast 8 months before data analysis in February 2012, and had measurabledisease at baseline and one of the following: at least 1 on-treatmentscan or clinical evidence of disease progression or death. ^(†)CRdenotes complete response, MEL melanoma, NSCLC non-small cell lungcancer, ORR objective response rate, PFSR progression-free survivalrate, PR partial response, RCC renal cell cancer, SD stable disease, nnumber of patients. ^(‡)Objective response rates ({[CR + PR] ÷ n} × 100)have been calculated based on confirmed responses with confidenceintervals calculated using the Clopper-Pearson method. Individualpatient responses were adjudicated per RECISTS v1.0 with modification(see Topalian et al., 2012b). ^(§)Progression-free survival rate was theproportion of patients who did not progress and were alive at 24 weekscalculated by the Kaplan-Meier methodology with confidence intervalsusing the Greenwood method. ^(¶)One CR. **One NSCLC patient who wastreated at the 3 mg/kg dose level had an initial evaluation ofprogressive disease, subsequently had a PR, and was classified as aresponder.

TABLE 2 Duration of Objective Responses to BMS-936558* as Assessed toFebruary 2012 No. of Tumor Dose Patients Type (mg/kg) with OR Durationof Response (months)^(†) MEL 0.1 4 7.5+, 5.6+, 5.6, 5.6 0.3 3 3.8+,2.1+, 1.9+ 1 8 24.9+, 22.9, 20.3+, 19.3+, 18.4+, 7.6+, 5.6+, 5.3+ 3 722.4+, 18.3+, 15.2+, 12.9, 11.1, 9.3, 9.2+ 10 4 24.6+, 23.9+, 18.0+,17.0 NSCLC^(§) 1 1 9.2+ 3 6 30.8+, 7.6+, 5.5+, 3.7+, 1.9+, NA^(‡) 10 714.8+, 7.6+, 7.3+, 6.7, 4.2, 3.7+, 3.7 RCC 1 4 17.5+, 9.2+, 9.2, 5.6+ 105 22.3+, 21.7+, 12.9, 12.0, 8.4 *MEL denotes melanoma, NA notapplicable, NSCLC non-small cell lung cancer, RCC renal cell cancer.^(†)Time from first response to time of documented progression, death,or for censored data, time to last tumor assessment. ^(‡)One patient wastreated beyond an initial evaluation of progressive disease andsubsequently had a PR; this patient was classified as a responder forthe purposes of calculating response rates by RECIST v1.0 but was noteligible for calculation of duration of response.

Data Analyzed as of March 2013

Objective responses were observed in patients with NSCLC (17%), MEL(31%), and RCC (29%), but not with CRC or CRPC. Responses were observedacross all nivolumab doses tested; response rates in NSCLC patientsreceiving 1 mg/kg, versus 3 or 10 mg/kg, appeared to be decreased (3%,versus 24% and 20%, respectively) (Tables 3 and 4). Thirteen of 270patients (4.8%) with responding histologies had unconventional responsepatterns that did not meet RECIST criteria (e.g., persistent reductionin target lesions in the presence of new lesions or regression followinginitial progression) (Wolchok et al, 2009). Additional patientsmanifested SD for 24 weeks or longer (10% NSCLC, 7% MEL, 27% RCC).Sustained survival, reflected by 1- and 2-year landmark OS rates, wasnoted in each of the responding populations as follows: NSCLC, 42% and14%; MEL, 62% and 43%; and RCC, 70% and 50% (Table 3). Median OS of 9.6months for lung cancer (9.2 and 10.1 months for squamous andnon-squamous NSCLC histologies, respectively), 16.8 months for MEL, andmore than 22 months for RCC, were observed; median PFS was 2.3 months inNSCLC, 3.7 months in MEL, and 7.3 months in RCC; and the median durationof response was 74, 104, and 56 weeks, respectively (Table 4). Among 16responders who discontinued therapy for reasons other than diseaseprogression and were followed for at least 24 weeks, 13 (81%) remainedin response at the time of analysis (FIG. 8).

TABLE 3 Clinical Activity of Nivolumab in the Efficacy Population (N =306)* as Assessed to March 2013 Stable Disease ORRs^(†) No. of MedianNo. of patients/ Median Median Overall Survival patients/total no.Duration of total no. of patients Progression- Overall Rate, % (95% CI);of patients (%) Response^(‡), (%) [95% CI] free Survival, Survival,patients at risk, n Tumor Type [95% CI] wk (range) ≧24 wk ≧48 wk mo (95%CI) mo (95% CI) 1 yr 2 yr Non-small cell 22/129 (17.1)  74.0 13/129(10.1)  6/129 (4.7)  2.3 9.6 42 14 (4, 24); 5 lung cancer** [11.0, 24.7](6.1+, 133.9+) [5.5, 16.6] [1.7, 9.8]  (1.9, 3.7) (7.8, 12.4) (33, 51);43 Squamous  9/54 (16.7) NR^(§) 8/54 (14.8) 3/54 (5.6) 3.7 9.2 39 ¶ [7.9, 29.3] (16.1, 133.9+) [6.6, 27.1] [1.2, 15.4] (1.8, 7.2) (7.3,12.5) (25, 53): 16 Non- 13/74 (17.6) 63.9 5/74 (6.8)  3/74 (4.1) 2.010.1  43 ¶ squamous  [9.7, 28.2] (6.1+, 74.0+)  [2.2, 15.1] [0.8, 11.4](1.8, 3.6) (7.2, 13.7) (31, 54): 26 Melanoma 33/107 (30.8)  104   7/107(6.5)  4/107 (3.7)  3.7 16.8  62 43 (32, 53); 26 [22.3, 40.5] (18.4,117.0+) [2.7, 13.0] [1.0, 9.3]  (1.9, 9.1) (12.5, 31.6)  (53, 72); 55Kidney cancer 10/34 (29.4) 56.1 9/34 (26.5) 2/34 (5.9) 7.3 >22^(# )  7050 (31, 70); 8  [15.1, 47.5] (36.6, 126.7+) [12.9, 44.4]  [0.7, 19.7] (3.7, 12.9) (13.6, NE{circumflex over ( )}) (55, 86); 23 *No objectiveresponses were seen in 19 patients with colorectal cancer or 17 patientswith castration-resistant prostate cancer. ^(†)Objective response rates({[CR + PR] ÷ n} × 100) have been calculated based on confirmedresponses with confidence intervals calculated using the Clopper-Pearsonmethod. Individual patient responses were adjudicated per RECIST v1.0with modification (see Methods S1 and study protocol, NEJM.org).^(‡)Time from first response to time of documented progression, death,or for censored data (denoted by “+”), time to last tumor assessment.**Among 129 patients with non-small-cell lung cancer, 1 had an unknownhistology and did not show an objective response. Others had squamous ornon-squamous histologies, as indicated. ^(§)NR, not reached; the timepoint at which the probability that responders progress drops below 50%has not been reached due to insufficient number of events and/orfollow-up. ¶ Insufficient period of follow-up. ^(∥)1 CR was noted inmelanoma, and 1 CR in kidney cancer. ^(#)The median overall survival wasnot reached at 22 months, the longest time to death so far in patientswith kidney cancer in this trial. {circumflex over ( )}NE, notestimable.

TABLE 4 Clinical Activity of Nivolumab by Dose Level as Assessed toMarch 2013 ORRs* No. of Median Stable Disease Median patients/totalDuration of No. of patients/total no. of Progression- Median OverallTumor Dose, no. of patients (%) Response wk patients (%) [95% CI] freeSurvival, Survival, mo Type mg/kg [95% CI] (range)^(†) ≧24 wk ≧48 wk mo(95% CI) (95% CI) Non-small All 22/129 (17.1)  74.0 13/129 (10.1)  6/129(4.7)  2.3 (1.9, 3.7) 9.6 (7.8, 12.4) cell doses [11.0, 24.7]  (6.1+,133.9+) [5.5, 16.6] [1.7, 9.8]  lung cancer 1 1/33 (3.0)  63.9 5/33(15.2) 2/33 (6.1) 1.9 (1.8, 3.6) 9.2 (5.6, 11.1) [0.1, 15.8] (63.9,63.9)  [5.1, 31.9] [0.7, 20.2] 3 9/37 (24.3) NR^(‡) 3/37 (8.1)  2/37(5.4) 1.9 (1.7, 7.3) 14.9 (9.5, NE{circumflex over ( )})  [11.8, 41.2] (16.1+, 133.9+)  [1.7, 21.9] [0.7, 18.2] 10 12/59 (20.3)  83.1 5/59(8.5)  2/59 (3.4) 3.6 (1.9, 3.8) 9.2 (5.2, 12.4) [11.0, 32.8]  (6.1+,117.1+) [2.8, 18.7] [0.4, 11.7] Squamous All 9/54 (16.7) NR 8/54 (14.8)3/54 (5.6) 3.7 (1.8, 7.2) 9.2 (7.3, 12.5) doses [7.9, 29.3] (16.1,133.9+) [6.6, 27.1] [1.2, 15.4] 1 0 0  4/15 (26.7) 1/15 (6.7) 1.9 (1.8,7.5) 8.0 (2.6, 13.3) [7.8, 55.1] [0.2, 31.9] 3 4/18 (22.2) NR 1/18(5.6)  1/18 (5.6)  3.8 (1.7, 14.9) 9.5, (6.7, NE)  [6.4, 47.6] (16.1,133.9+) [0.1, 27.3] [0.1, 27.3] 10 5/21 (23.8) 83.1 3/21 (14.3) 1/21(4.8) 4.1 (1.8, 7.6) 10.5 (7.8, 12.5)  [8.2, 47.2] (16.1, 117+)  [3.0,36.3] [0.1, 23.8] Non- All 13/74 (17.6)  63.9 5/74 (6.8)  3/74 (4.1) 2.0(1.8, 3.6) 10.1 (7.2, 13.7)  squamous doses [9.7, 28.2] (6.1+, 74.0+) [2.2, 15.1] [0.8, 11.4] 1 1/18 (5.6)  63.9 1/18 (5.6)  1/18 (5.6) 1.8(1.7, 3.6) 9.9 (5.6, 22.7) [0.1, 27.3] (63.9, 63.9)  [0.1, 27.3] [0.1,27.3] 3 5/19 (26.3) 74.0 2/19 (10.5) 1/19 (5.3)  1.8 (1.7, 12.5) 18.2(10.3, 18.2) [9.1, 51.2] (24.3, 74.0+)  [1.3, 33.1] [0.1, 26.0] 10 7/37(18.9) NR 2/37 (5.4)  1/37 (2.7) 2.3 (1.9, 3.8) 7.4 (4.6, 12.4) [8.0,35.2] (6.1+, 65.7+)  [0.7, 18.2] [0.1, 14.2] Melanoma^(§) All 33/107(30.8)  104    7 (6.5)   4 (3.7) 3.7 (1.9, 9.1) 16.8 (12.5, 31.6) doses[22.3, 40.5]  (18.4, 117.0+) [2.7, 13.0] [1.0, 9.3]  0.1^(¶) 6/17 (35.3)NR 0 0 3.6 (1.7, 9.1) 16.2 (8.6, NE)  [14.2, 61.7]  (24.1, 48.7+) 0.3^(¶) 5/18 (27.8) NR  1 (5.6) 0 1.9 (1.8, 9.3) 12.5 (7.7, NE)  [9.7,53.5] (18.4, 66.3+)  [0.1, 27.3] 1 11/35 (31.4)  104   5/35 (14.3)  4/35(11.4)  9.1 (1.8, 24.7) 25.3 (14.6, NE)  [16.9, 49.3]  (32.4, 108.1+)[4.8, 30.3] [3.2, 26.7] 3 7/17 (41.2) 75.9 1/17 (5.9)  0  9.7 (1.9,16.4) 20.3 (8.2, NE)  [16.9, 67.1]  (40.1+, 115.4+)  [0.1, 28.7] 10 4/20(20.0) 112   0 0  3.7 (1.7, 20.50) 11.7 (7.2, 37.8)  [5.7, 43.7] (73.9,117+)  Kidney All 10/34 (29.4)  56.1 9/34 (26.5) 2/34 (5.9)  7.3 (3.7,12.9) >22^(#) (13.6, NE)   cancer^(§) doses [15.1, 47.5]  (36.6, 126.7+)[12.9, 44.4]  [0.7, 19.7] 1 5/18 (27.8) 56.1 4/18 (22.2) 1/18 (5.6)  4.7(1.9, 10.9) NR (17.9, NE) [9.7, 53.5] (40.1, 76.1+)  [6.4, 47.6] [0.1,27.3] 10 5/16 (31.3) 56.1 5/16 (31.3) 1/16 (6.3)  8.0 (3.7, 14.0) 18.8(11.6, NE)  [11.0, 58.7]  (36.6, 126.7+) [11.0, 58.7]  [0.2, 30.2]*Objective response rates ({[CR + PR] ÷ n} × 100) have been calculatedbased on confirmed responses with confidence intervals calculated usingthe Clopper-Pearson method. Individual patient responses wereadjudicated per RECIST v1.0 with modification (see Methods S1 and studyprotocol, NEJM.org). ^(†)Time from first response to time of documentedprogression, death, or for censored data (denoted by “+”), time to lasttumor assessment. ^(‡)NR, not reached; the time point at which theprobability that responders progress drops below 50% has not beenreached due to insufficient number of events and/or follow-up. ^(§)1 CRwas noted in melanoma, and 1 CR in kidney cancer. ^(¶)Five patients withtumor progression were dose-escalated from 0.1 to 1.0 mg/kg, and sixfrom 0.3 to 1.0 mg/kg. None of these patients responded to therapy.^(#)The median overall survival was not reached at 22 months, thelongest time to death so far in patients with kidney cancer in thistrial. {circumflex over ( )}NE, not estimable.

Example 8 Correlation Between Membranous PD-L1 Expression and Anti-PD-1Response

IHC staining of PD-L1 was performed on pretreatment formalin-fixedparaffin-embedded (FFPE) tumor specimens using the murine anti-humanPD-L1 mAb 5H1 (Dong et al., 2002) in a standard IHC protocol (Taube etal., 2012; Supp. Materials). Briefly, 5 μm-FFPE sections mounted onglass slides were deparaffinized in xylene and antigen retrieval wasperformed using a Tris-EDTA buffer, pH 9.0 at 120° C. for 10 min in aDecloaking Chamber (Biocare Medical). Endogenous peroxidase, biotin andproteins were blocked (CAS system K1500, Dako; Avidin/biotin BlockingKit, SP-2001, Vector Laboratories; Serotec Block ACE), and the primary5H1 Ab was added at a concentration of 2 μg/ml and allowed to incubateat 4° C. for 20 h. Secondary Ab (biotinylated anti-mouse IgG1, 553441BD) was applied at a concentration of 1 μg/ml for 30 min at roomtemperature (RT). The signal was then developed with amplificationaccording to the manufacturer's protocol (CAS system K1500, Dako).Sections were counterstained with hematoxylin, dehydrated in ethanol andcleared in xylene, and a coverslip was applied.

The percentage of tumor cells exhibiting cell surface staining for PD-L1was scored by two independent pathologists who were blinded to treatmentoutcomes. PD-L1 positivity was defined per specimen by a 5% expressionthreshold (Taube et al., 2012; Thompson et al., 2006), and in cases withmultiple specimens, if any specimen met this criterion. A Fisher's exacttest was applied to assess the association between PD-L1 expression andOR, noting, however, that this analysis was based in part on optionalbiopsies from a non-random subset of the population and testing of astatistical hypothesis was not pre-specified.

Sixty-one pretreatment tumor specimens from 42 patients (18 MEL, 10NSCLC, 7 CRC, 5 RCC, and 2 CRPC) were analyzed for tumor cell surfacePD-L1 expression (FIG. 8). Biopsy specimens from 25 of 42 patients werepositive for PD-L1 expression by IHC. A Fisher's exact test was appliedto assess the association between PD-L1 expression and OR in a post-hocanalysis. Among the 42 surface-PD-L1⁺ patients, 9 (36%) achieved an OR,whereas among 17 patients with PD-LF tumors, none achieved an OR (FIG.8A). Thus, in a subset of patients cell surface expression of PD-L1 ontumor cells in pretreatment biopsies is associated with an increasedrate of OR among patients treated with BMS-936558, while no patientswith documented PD-L1-negative tumors experienced an OR. These dataindicate that tumor PD-L1 expression is a molecular marker that canenable patient selection for anti-PD-1 immunotherapy.

Example 9 Isolation of Rabbit mAbs that Detect Membranous hPD-L1 Antigenin FFPE Tissues

Rabbit Abs against human PD-L1 polypeptide were prepared by Epitomics,Inc. (Burlingame, Calif.) by immunization of rabbits using a recombinanthuman PD-L1 fusion protein. Antiserum titers were evaluated usingstandard direct ELISA with the hPD-L1 antigen, and cell ELISA usingtransfected cells overexpressing hPD-L1. These Abs were also screenedfor their ability to bind to PD-L1 by IHC assay of FFPE tissue sections.The rabbit with the highest Ab titer was selected for splenectomy.Lymphocytes isolated from the spleen were fused to myeloma cells in40×96-well plates, and screened by ELISA against the immunizing PD-L1antigen and by cell ELISA against cells overexpressing hPD-L1. Positiveclones were expanded into 24-well plates, and confirmatory screens wereconducted by direct ELISA and cell ELISA. The supernatants (sups) ofclones that were specific to the screening antigen were re-screened byIHC.

A set of mouse anti-hPD-L1 mAbs were also produced by immunization ofmice using a protocol similar to that described above for the rabbitmAbs.

Out of a total of 185 multiclones from both rabbit and mouseimmunizations screened, only ten rabbit multiclone Abs specificallydetected the membranous form of hPD-L1. None of the purified mousesubclones were found to specifically detect cell surface hPD-L1. Sixtysubclones from the top five rabbit multiclones (designated Nos. 13, 20,28, 29 and 49, each comprising 12 subclones) were screened initially byIHC on FFPE low density tissue microarrays (TMAs), followed byconfirmation and specificity verification in narrowed 25 subclones.Rabbit IgG was used as a negative isotype control, and mAb 5H1 (Dong etal., 2002) was used as the positive control. Specificity was furtherverified by antigen preabsorption assay. Through two rounds of IHC, thefollowing 15 purified subclones were selected as the most promising Absin terms of specificity and intensity of staining: 13-1, 13-3, 13-7,13-8; 20-5, 20-7, 20-12, 20-6; 28-1, 28-8, 28-12; 29-8; 49-5, 49-7 and49-9 Immunoreactivity data on these selected Abs are summarized in Table5.

TABLE 5 Immunoreactivity of Rabbit Anti-hPD-L1 mAbs Specific StainingNon-Specific staining Pos. vs Neg. Intensity Range on BackgroundStaining on Antibody Staining on Tissues^(†) (Very High, Tissues (High,Medium, Name PD-Ll Cells* High, Medium, Low) Low) 13-1 Pos Low to VeryHigh None 13-3 Pos Low to High None 13-7 Pos Low to High None 13-8 PosLow to High None 20-5 Pos Low to Very High High 20-6 Pos Low to HighMedium 20-7 Pos Low to High Medium 20-12 Pos Low to High Medium 28-1 PosLow to Very High None 28-8 Pos Medium to Very High None 28-10 Pos Mediumto Very High Low 28-12 Pos Low to Very High None 49-5 Pos Low to HighNone 49-7 Pos Low to High Low 49-9 Pos Low to High None 5H1 Pos High toVery High None Rb IgG Neg Neg None *PD-L1 stably transfected CHO cellsvs. CHO-S control; ^(†)PD-L1 positive tissues included placenta and onenon-small cell lung cancer; Detection up to “very high” expressionsuggests better sensitivity at detecting membranous PD- L1.

Additional assays were performed to further characterize the purified Abclones, including determining binding affinity and cross-competitionamong the Abs (to identify overlapping versus different epitope regions)by surface plasmon resonance. All the Abs exhibited high bindingaffinity (K_(D)<10⁻⁹ M). These 15 purified clones were also re-screenedby IHC on FFPE low density TMA or regular sections against various celland tissue types known to be positive or negative for cell surfaceexpression of PD-L1. Rabbit IgG was used as the isotype control, and mAb5H1 was used as the positive control. At high concentration (10 μg/ml),clones 28-x and 49-x displayed low to moderate levels of backgroundstaining in tissues, while clones 13-x exhibited no background staining,which suggests that the 13-x clones have a wider dynamic range. The 20-xclones displayed various degree of background staining which wasprimarily cytoplasmic and diffuse. The clone with most robust detectionspecifically of membranous PD-L1, rabbit clone 28-8 (K_(D)=100 p M, asdetermined by SPR), was selected as the lead Ab for subsequent IHCassays. MAbs 28-1, 28-12, 20-12 and 29-8 had K_(D) values of 130 pM, 94pM, 160 pM and 1200 pM, respectively. The sequences of the heavy chainvariable (V_(H)) and light (kappa) chain variable (V_(κ)) regions of mAb28-8 are set forth in SEQ ID NOs. 35 and 36, respectively. The 28-8 Abwas shown to recognize a different epitope from mouse mAb 5H1, based onSPR analysis. Clones 28-1, 28-12, 29-8 and 20-12 were the next best Absin terms of robust detection of membranous PD-L1 in FFPE tissues.Although mAb 13-1 had the best specificity in terms of detection ofmembranous PD-L1, the maximal detection level was lower than that of theother lead Abs. Western blotting was also performed with plus/minusantigen competition to verify the specificity of the top selected Absfor PD-L1.

The binding of mAbs 5H1 and 28-8 to membranous PD-L1 in FFPE test tissuesamples comprising tumor cells and tumor-infiltrating inflammatory cellsfrom different types of tumors was compared. Membranous PD-L1 expressionwas evaluated using the histoscore method performed by 2 independentpathologists. Four NSCLC, 2 MEL, and 2 RCC tumors were stained with 28-8at 2 μg/ml and 5H1 at 5 μg/ml. The data are tabulated in Table 6, andshown graphically in FIG. 9. The rabbit mAb 28-8 showed better detection(higher histoscores) for 7 out of 10 samples using 2.5-fold less Ab, andin only one sample was the histoscore for 5H1 slightly higher than formAb 28-8.

TABLE 6 Comparison of mAbs 28-8 and 5H1 by histoscore analysisHistoscore Histoscore Tissue Sample I.D. Average (5H1) Average (28-8)NSCLC 1080754B 245 261 NSCLC 1080766B 103 130 NSCLC 1080790 40 37 NSCLC1080993B 12 16 Mel T030668 98 113 Mel T980744 98 123 Mel 1168657B 0 0Mel T980747 1 9 RCC 1164619B 4 4 RCC 1167809B 108 125

Taube et al. (2012) demonstrated by flow cytometry on cultured cellsthat mAb 5H1 bound to the cell surface, and the specificity of bindingto PD-L1 was confirmed using a PD-L1 fusion protein to competitivelyblock binding of the 5H1 mAb to tissue sections. These authors alsocompared 5H1 with a rabbit polyclonal anti-hPD-L1 Ab, 4059, previouslydescribed by Gadiot et al. (2011), and found that whereas 5H1 showed acell surface staining pattern on FFPE samples, pAb 4059 demonstrateddiffuse cytoplasmic staining. Further, when 5H1 was compared to pAb 4059by western blot analysis, Ab 4059 bound to multiple proteins in lysatesof melanoma cells in addition to a 50 kDa protein corresponding to theexpected mass of glycosylated PD-L1, in contrast to 5H1 whichspecifically detected the 50 kDa band of glycosylated PD-L1 (Taube etal., 2012). Contrary to the results of Taube et al. (2012) and theresults disclosed herein (summarized in Table 7), Gadiot et al. (2011)reported that mAb 5H1 produced a high level background staining in FFPEtissue samples, whereas they found that pAb 4059 produced satisfactoryspecific staining of PD-L1 in FFPE samples. Thirteen other Abs tested byGadiot et al. (2011) either did not stain FFPE tissues, gave a highbackground staining, or were not blocked by PD-L1 fusion proteincompetition, highlighting the difficulties in obtaining anti-PD-L1 Absthat bind specifically to PD-L1 in FFPE tissues.

In the present study, an automated IHC assay (see Example 10) was usedto evaluate the binding of several commercially available anti-PD-L1 Absand 5H1 (Dong et al., 2002) to FFPE tissue samples containing variouscells expressing PD-L1. The results, summarized in Table 7, show thatnone of the commercially available Abs tested specifically recognizedmembranous PD-L1 expression in human tissues known to express PD-L1, orto clearly distinguish CHO cells expressing PD-L1 versus theuntransfected parent CHO cells that did not express PD-L1. The inabilityof the polyclonal Ab (pAb) 4059 to bind specifically recognizedmembranous PD-L1 is consistent with the findings of Taube et al. (2012).The binding of 28-8 was similar to that of 5H1 in this assay, thoughhistoscore analysis suggests that 28-8 performs better than 5H1.

TABLE 7 Binding of mAbs to FFPE Samples Containing PD-Ll-expressingcells Clone No. Pos. vs. Neg. Human Antibody (mAb)/Catalog Staining onPositive Source Types No. (pAb) PD-L1 Cells* Tissues^(†) MBL mAb 27A2Failed Failed BioLegend mAb 29E.2A3 Failed Failed eBiosciences mAb M1H1No Staining No staining Collaborator mAb 5H1 Passed Passed ProSci pAb4059 Failed Failed LifeSpan pAb LS-B480/0604 Failed Failed BioSciencesBristol-Myers mAb 28-8 Passed Passed Squibb *PD-L1 stably transfectedCHO cells vs. parent CHO-S negative control; ^(†)PD-L1 positive tissuesincluded tonsil and/or thymus; mAb, mouse monoclonal Ab; pAb, rabbitpolyclonal Ab.

Example 10 Development of Automated IHC Protocol for Assessing PD-L1Expression

An automated IHC protocol was developed to assay PD-L1 expression inFFPE specimens. Tissue sections (4 μm) were mounted on slides,deparaffinized in an autostainer (Leica) by soaking twice for 5 min inxylene, and re-hydrated by soaking twice for 2 min each time in 100%EtOH, twice in 95% (v/v) EtOH, once in 70% (v/v) EtOH, and once inde-ionized water (dH₂O). Antigen retrieval was performed using adecloaking chamber (Biocare Medical Decloaking Chamber Plus) and Dako pH6 buffer, heated to 110° C. (P1) for 10 min, then moved to the next step(P2 FAN ON at 98° C.; FAN OFF at 90° C.). The slides were cooled at roomtemperature (RT) for 15 min and rinsed with water for about 1 min.

Reagents were set up on an autostainer (BioGenex i6000), and the tissuearea was defined using a pap pen. The IHC assay, run using theautostainer in research mode, comprised the following steps:neutralizing endogenous peroxidase using the Peroxidase Block (Leica)for 10 min, followed by rinsing 3 times with IHC wash buffer (Dako);applying Protein Block (Leica) to the slides, and incubating for 10 minat RT, followed by washing 3 times with wash buffer; applying theprimary Ab to the slides (2 μg/ml) and incubating for 1 h at RT,followed by washing 6 times with wash buffer; adding Post Primary Block(NovoLink Kit) to the slides and incubating for 30 min, followed bywashing 6 times with wash buffer; adding NovoLink Polymer (NovoLink Kit)to the slides and incubating for 30 min, followed by washing 6 timeswith wash buffer; adding the DAB chromogen substrates (NovoLink Kit) anddeveloping for 3 min, followed by rinsing 5 times with dH₂O at RT;counterstaining with hematoxylin (NovoLink Kit) for 1 min at RT,followed by washing 3 times with dH₂O for 5 times at RT. The primary Abwas selected from the rabbit anti-PD-L1 Abs shown in Table 4; mAb 28-8was the preferred Ab. As a negative control, rabbit IgG (Dako) was used.The tissue sections were dehydrated using a Leica autostainer by washingonce for 2 min in 70% EtOH, twice for 2 min in 95% EtOH, and three timesfor 2 min in 70% EtOH, then cleared by washing three times for 5 min inxylene. The sections were permanently mounted with permount to theslide, covered with a coverslip, and transferred to a chemical hood todry.

Example 11 Design of Phase 1 Clinical Study on Anti-PD-L1 Antibody

Study Design

A phase 1 study was conducted to assess the safety and tolerability ofBMS-936559 (also referred to herein and in U.S. Pat. No. 7,943,743 as12A4) in patients with selected advanced solid tumors. Secondaryobjectives included initial assessment of the antitumor activity ofBMS-936559 and pharmacokinetic evaluation. Pharmacodynamic measures wereincluded under exploratory objectives. Patients were treated in 6-weekcycles of BMS-936559 administered as a 60-minute intravenous infusionevery 2 weeks on days 1, 15, and 29 of each cycle. Patients continuedtreatment for up to 16 cycles unless they experienced unacceptabletoxicity, disease progression, or withdrew consent. In some patients whowere clinically stable, treatment beyond initial disease progression waspermitted until further progression was confirmed.

Dose Escalation

Patients with advanced NSCLC, MEL, CRC, RCC, ovarian (OV), gastric (GC),breast (BC), and pancreatic (PC) carcinoma were eligible to enroll.Using an accelerated titration design, safety was assessed at doses of0.3, 1, 3, and 10 mg/kg. One patient was enrolled in each successivecohort until there was a ≧grade 2 drug-related AE during cycle 1. Twoadditional patients were then enrolled at that dose level and the studywas transitioned to a standard 3+3 design. Intra-patient dose escalationor de-escalation was not permitted. The maximum tolerated dose (MTD) wasdefined as the highest dose where less than one-third of patients had adose-limiting toxicity.

Cohort Expansion

Initially, 5 expansion cohorts (n=16/cohort) were enrolled at 10 mg/kgfor patients with NSCLC, MEL, RCC, OV, and CRC. Based on initial signalsof activity, additional expansion cohorts (up to n=16/cohort) wereenrolled for MEL (at 1.0 and 3.0 mg/kg), NSCLC (squamous or nonsquamoushistology cohorts randomized to 1, 3, or 10 mg/kg), and at 10 mg/kg forPC, BC, and GC.

Patients

Patients were required to have documented advanced NSCLC, MEL, RCC, OV,CRC, PC, GC, or BC, and have failed at least one prior tumor-appropriatetherapy for advanced/metastatic disease (except for PC or GC patientswho could be treatment-naïve). Other inclusion criteria included age ≧18years, life expectancy ≧12 weeks, Eastern Cooperative Oncology Groupperformance status of ≦2, measurable disease as defined by RECIST v1.0,and adequate hematologic, hepatic, and renal function. Patients withtreated brain metastases were allowed, if stable for at least 8 weeks.Major exclusion criteria included a history of autoimmune disease orother diseases requiring systemic steroids or immunosuppressivemedication, prior therapy with T cell-modulating Abs (includinganti-PD-1, anti-PD-L1 and anti-CTLA-4), history of HIV, or activehepatitis B or C.

In this ongoing study, 207 patients with NSCLC (n=75), MEL (n=55), CRC(n=18), RCC (n=17), OV (n=17), PC (n=14), GC (n=7), or BC (n=4) weretreated with BMS-936559 during a 34-month period and are included in thesafety data. Efficacy was characterized in 160 response-evaluablepatients. The baseline demographic characteristics of the total andresponse-evaluable patient populations were very similar (Brahmer etal., 2012). Among treated patients, 86% had received prior chemotherapyand 28% immunologic or biological therapy. Prior therapies by tumor typeincluded immunotherapy (56%) and B-RAF inhibitor (9%) in patients withMEL; platinum-based chemotherapy (95%) and tyrosine kinase inhibitors(TKIs; 41%) in patients with NSCLC; and nephrectomy (94%),anti-angiogenic therapy (82%), and immunotherapy (41%) in patients withRCC (see Brahmer et al., 2012, for more details on patientpre-treatments).

Statistical Analysis

All 207 patients commencing treatment as of the date of analysis wereused for summaries of baseline characteristics and AEs. The efficacypopulation consisted of 160 response-evaluable patients who initiatedtreatment at least 7 months before the date of analysis. AEs were codedusing MedDRA v14.1. Individual best overall responses were derived fromradiographic scan measurements according to modified RECIST v1.0. ORswere confirmed by at least one sequential tumor assessment. Additionaldetails regarding statistical methods are provided in Brahmer et al.(2012).

Example 12 Safety Evaluations on Patients Treated with Anti-PD-L1Antibody

Safety evaluations (clinical examination and laboratory assessments)were conducted on all treated patients at baseline and regular intervals(weekly during cycle 1 and biweekly thereafter). AE severity was gradedbased on the NCI CTCAE, v3.0. Disease assessment via computed tomography(CT) scans or magnetic resonance imaging was performed at baseline andprior to each treatment cycle.

A MTD was not reached up to the highest tested dose of 10 mg/kg ofBMS-936559. The median duration of therapy was 12 weeks (range 2.0-111.1weeks). A relative dose intensity of ≧90% was achieved in 86% ofpatients. Twelve of 207 patients (6%) discontinued treatment due to aBMS-936559-related AE (see Brahmer et al., 2012, for details).

AEs regardless of causality (any grade) were reported in 188 of 207patients. Investigator-assessed BMS-936559-related AEs were noted in 126of 207 (61%) patients. The most common drug-related AEs were fatigue,infusion reactions, diarrhea, arthralgia, rash, nausea, pruritus, andheadache. Most events were low grade with BMS-936559-related grade 3-4events noted in 19 of 207 (9%) patients (Brahmer et al., 2012). Thespectrum, frequency, and severity of BMS-936559-related AEs were similaracross the dose levels, with the exception of infusion reactions.Drug-related AEOSIs, with potential immune-related etiologies, wereobserved in 81 of 207 (39%) of the patients and included rash,hypothyroidism, hepatitis, and single cases each of sarcoidosis,endophthalmitis, diabetes mellitus, and myasthenia gravis (Brahmer etal., 2012). These AEs were predominantly grade 1-2 and generallyreversible with treatment interruption or discontinuation. Notably, 9patients were treated with corticosteroids for the management of AEs.AEs improved or resolved in all patients. Furthermore, 4 of these 9patients maintained disease control despite treatment withcorticosteroids. Endocrine AEs were managed with replacement therapy andpatients reinitiated treatment with BMS-936559 at the discretion of thetreating physician. Infusion reactions were observed in 21 of 207 (10%)patients, predominantly at 10 mg/kg. They were grade 1-2 with theexception of one grade 3 event at 10 mg/kg. Infusion reactions weregenerally rapidly reversible with antihistamines and antipyretics and,in some cases, corticosteroids. A prophylactic regimen withantihistamines and antipyretics was implemented during the study.Patients with grade 1-2 infusion reactions were able to continuetreatment with BMS-936559 with prophylactic antihistamines andantipyretics and at a reduced infusion rate. BMS-936559-related seriousAEs occurred in 11 of 207 (5%) patients. As of the data analysis date,45 patients (22%) had died (Brahmer et al., 2012); no drug-relateddeaths were observed.

Example 13 Pharmacokinetics/Pharmacodynamics Analyses on Anti-PD-L1Antibody

For PK analyses, serial blood samples were collected and serumconcentrations of BMS-936559 were quantified by ELISA. Peripheral bloodmononuclear cells were isolated from patients at baseline and followingone treatment cycle to assay PD-L1 RO by BMS-936559 on circulatingCD3-positive T-cells via flow cytometry (Brahmer et al., 2010).

Serum concentrations of BMS-936559 increased in a dose-dependent mannerfrom 1-10 mg/kg (n=131). Geometric mean area under the curve (0-14 days)for the 1, 3, and 10 mg/kg dose levels were 2210, 7750, and 36620μg/mL·hr (coefficient of variation [CV] 34-59%), respectively; geometricmean peak concentrations at these dose levels were 27, 83, and 272 μg/mL(CV 30-34%), respectively, after the first dose. The half-life ofBMS-936559 was estimated from population pharmacokinetic data asapproximately 15 days. PD-L1 RO on CD3-positive peripheral bloodlymphocytes was assessed in 29 MEL patients at the end of 1 cycle oftreatment, at BMS-936559 doses from 1-10 mg/kg. Median RO exceeded 65%for all groups (Brahmer et al., 2012).

Example 14 Antitumor Efficacy Exhibited by Anti-PD-L1 Antibody

One-hundred and sixty patients out of the 207 treated were evaluable forresponse by February 2012 and included those with NSCLC, MEL, CRC, RCC,OV, and PC, but not patients with GC or BC. Clinical activity wasobserved at all doses ≧1 mg/kg (Brahmer et al., 2012). ORs (confirmedcomplete [CR] or partial [PR] responses) were observed in patients withMEL, NSCLC, RCC, and OV (Table 8), as illustrated by representativespider plots and CT scans (FIGS. 10-13), and many ORs were also durable(Table 9). Four additional patients had a persistent reduction in targetlesions in the presence of new lesions, consistent with an“immune-related” pattern of response; however, for the purpose ofcalculating response rates, they were not categorized as responders.Antitumor responses and/or prolonged stable disease (SD) were observedin patients with a variety of prior therapies received. ORs wereobserved even in patients with an extensive burden of metastaticdisease.

In patients with MEL, there were 9 ORs across the 1, 3, and 10 mg/kgdose levels, with response rates of 6%, 29%, and 19%, respectively.Three MEL patients achieved a CR. All 9 MEL patients who experienced anOR started treatment ≧1 year prior to data analysis; of these 5 had aresponse duration ≧1 year. Additionally 14 MEL patients (27%) had SDlasting ≧24 weeks. In patients with NSCLC, there were 5 ORs amongst the3 and 10 mg/kg dose levels, with response rates of 8% and 16%,respectively. There were ORs in patients with non-squamous (n=4) orsquamous histology (n=1). All 5 NSCLC responders started treatment ≧24weeks prior to data analysis; of these, 3 had responses lasting ≧24weeks. Six additional NSCLC patients had SD lasting ≧24 weeks. There was1 PR out of 17 patients with OV (6% response rate) and 3 patients (18%)with SD lasting ≧24 weeks, all at the 10 mg/kg dose. In patients withRCC, there were ORs in 2 of 17 (12%) patients treated at 10 mg/kg withresponses lasting 4 and 18 months, respectively. Seven additional RCCpatients had SD lasting ≧24 weeks.

TABLE 8 Clinical Activity of BMS-936559 in 160 Patients, ResponseEvaluable* ORO^(§) SD ≧ 24 wk PFSR** at Dose No. patients (%) No.patients (%) 24 wk (%) Tumor Type (mg/kg) n [95% CI] [95% CI] [95% CI]MEL 0.3 1 0 [0-98] 0 [0-98] N/A 1 18 1 (6) [0.1-27] 6 (33) [13-59] 39[16-61] 3 17 5 (29)^(†) [10-56] 3 (18) [4-43] 47 [21-72] 10 16 3(19)^(††) [4-46] 5 (31) [11-59] 44 [19-68] ALL MEL 52 9 (17) (8-30) 14(27) [16-41] 42 [28-56] NSCLC^(§) 1 11 0 [0-29] 0 [0-29] N/A Squamous 11 0 [0-98] 0 [0-98] N/A Non-Squamous 1 10 0 [0-31] 0 [0-31] N/A 3 13 1(8) [0.2-36] 1 (8) [0.2-36] 34 [7-60] Squamous 3 4 0 [0-60] 1 (25)[0.6-81] 50 [1-99] Non-Squamous 3 9 1 (11) [0.3-48] 0 [0-34] 25 [0-55]10 25 4 (16) [5-36] 5 (20) [7-41] 46 [25-67] Squamous 10 8 1 (13)[0.3-53] 2 (25) [3-65] 47 [10-83] Non-Squamous 10 17 3 (18) [4-43] 3(18) [4-43] 46 [20-72] ALL NCSLC 49 5 (10) [3-22] 6 (12) [5-25] 31[17-45] ALL Squamous 13 1 (8) [0.2-36] 3 (23.1) [5-54] 43 [15-71] ALLNon-Squamous 36 4 (11) [3-26] 3 (8) [2-23] 26 [10-42] OV 3 1 0 [0-98] 0[0-98] N/A 10 16 1 (6) [0.2-30] 3 (19) [4-46] 25 [4-46] ALL OV 17 1 (6)[0.1-29] 3 (18) [4-43] 22 [2-43] RCC 10 17 2 (12) [2-36] 7 (41) [18-67]53 [29-77] CI denotes Confidence intervals, MEL melanoma, RCC renal cellcarcinoma, NSCLC non-small cell lung cancer, OV ovarian cancer, RCCrenal cell carcinoma, N/A not applicable, ORR objective response rate(complete response + partial response), SD stable disease, and PFSRprogression-free survival rate. *Efficacy population consists ofresponse-evaluable patients who initiated treatment at least 7 monthsprior to the date of analysis and had measurable disease at a baselinetumor assessment and at least one of the following: an on-study tumorassessment, clinical progression, or death. ^(†)Includes two CRs.^(††)Includes one CR. ^(§)Objective response rates ({[CR + PR] ÷ n} ×100) are based on confirmed responses only, with confidence intervalscalculated using the Clopper-Pearson method. **Progression-free survivalrate was the proportion of patients who did not progress, and were aliveat 24 weeks, calculated by the Kaplan-Meier methodology, with confidenceintervals using the Greenwood method.

-   Individual patient responses were adjudicated per RECIST v1.0 with    modification (see study protocol in Brahmer et al. (2012) N Engl J    Med (submitted) for additional information).

TABLE 9 Duration of Objective Responses to BMS-936559* Tumor Dose No. ofPatients Type (mg/kg) with OR Duration of Response (months)^(†) MEL 1 16.9 3 5 23.5+, 22.9+, 16.2+, 4.1+, 3.5 10 4 24.6+, 23.9+, 18.0+, 17.0NSCLC 1 0 9.2+ 3 1 2.3+ 10 4 16.6+, 12.6+, 9.8, 3.5 RCC 10 2 18, 4 OV 101 1.3+ *MEL denotes melanoma, NSCLC non-small cell lung cancer, RCCrenal cell cancer, OV ovarian cancer. ^(†)Time from first response totime of documented progression, death, or for censored data (denoted by+), time to last tumor assessment.

Example 15 Design of Phase 1 Clinical Study on Anti-PD-1 PlusAnti-CTLA-4 in Advanced MEL Study Design

In this phase 1 study, successive cohorts of patients were treated withescalating doses of intravenous nivolumab and ipilimumab administeredconcurrently (concurrent regimen), and separately, two cohorts ofpatients treated previously with ipilimumab received nivolumab alone(sequenced regimen).

For the concurrent regimen, patients received during the inductionperiod nivolumab and ipilimumab every 3 weeks for 4 doses, followed bynivolumab alone every 3 weeks for 4 doses. Combined treatment wassubsequently continued during the maintenance period every 12 weeks forup to 8 doses. When both drugs were administered together, nivolumab wasadministered first. Within a cohort, nivolumab and ipilimumab doses werekept constant during the induction and maintenance periods. Thedose-limiting-toxicity evaluation period was through week 9. Tumorassessments were at weeks 12, 18, 24, and 36, and then every 12 weeksthereafter.

In the sequenced regimen, patients previously treated with ipilimumabprior to study entry received nivolumab every 2 weeks for up to 48doses. Nivolumab therapy was initiated within 4-12 weeks afteripilimumab monotherapy. Tumor assessments were at week 8 and then every8 weeks thereafter. Tumor responses were adjudicated using mWHO andimmune-related mWHO criteria for both regimens.

After completion of therapy, patients without confirmed diseaseprogression were followed for up to 2.5 years. Patients with CR, PR, orSD ≧24 weeks and subsequent disease progression could be retreated withthe original regimen. Safety evaluation was performed per protocol. Theseverity of AEs was graded according to the National Cancer InstituteCommon Terminology Criteria for Adverse Events, version 3.0. Diseaseassessment, using CT and/or MRI as appropriate, was performed perprotocol.

Dose Escalation and Cohort Expansion

The study was initially planned to evaluate the concurrent regimen usinga standard 3+3 design for the dose escalation phase, followed by cohortexpansion to a total of up to 16 patients at the maximum tolerated doseor the maximum administered dose. The dose-limiting toxicity (DLT)evaluation period for dose escalation was 9 weeks. No intra-patient doseescalation was allowed and patients who experienced DLT werediscontinued from therapy. Patients who withdrew from study during theDLT evaluation period for reasons other than drug-related toxicity couldbe replaced. The protocol was amended to permit expansion of anyconcurrent regimen cohort during dose escalation to N=up to 12 patients.Two sequenced-regimen cohorts (6 to 16 patients each) were later added;patients were treated with nivolumab (1 mg/kg or 3 mg/kg) after havingreceived prior ipilimumab.

Patients

Eligible patients were 18 years of age and had a diagnosis ofmeasurable, unresectable stage III or IV melanoma; Eastern CooperativeOncology Group performance status of 0-1, where 0 is asymptomatic and 1is mildly symptomatic; adequate organ function; and life expectancy of≧4 months. Patients with active, untreated central nervous systemmetastasis; history of autoimmune disease; prior therapy with T-cellmodulating antibodies (excluding ipilimumab for sequenced-regimencohorts); HIV; or hepatitis B or C were excluded.

In the sequenced-regimen cohorts, patients were required to havereceived 3 prior doses of ipilimumab, with the last dose administeredwithin 4-12 weeks of initiation of nivolumab. Patients with CR,progression with evidence of clinical deterioration, or a history ofhigh-grade AEs related to ipilimumab were excluded (see Wolchok et al.2013a, for details of protocol).

Eighty-six patients were treated between December 2009 and February2013, 53 with the concurrent regimen and 33 with the sequenced regimen.Baseline patient characteristics are detailed in Wolchok et al. (2013a).In the concurrent and sequenced regimens, 38% and 100% of patients,respectively, received prior systemic therapy. The majority of patientshad M1c disease and >30% had elevated serum lactate dehydrogenase (LDH).Most patients enrolled to the sequenced-regimen cohorts demonstratedradiographic progression (73%) with prior ipilimumab treatment.

PD-L1 Immunohistochemistry

Pre-treatment PD-L1 expression was measured by IHC in FFPE tumorspecimens using the rabbit anti-PD-L1 mAb, 28-8, and an automated assaydeveloped by Dako (Carpinteria, Calif.). Ab specificity was assessed bywestern blotting against recombinant PD-L1 protein and lysates fromPD-L1 expressing and non-expressing cell lines. An IHC assay with andwithout antigen competition and an assessment of staining patterns innormal human tissues was performed. Analytical sensitivity, specificity,repeatability, reproducibility, and robustness of theimmunohistochemistry assay were tested and met all pre-specifiedacceptance criteria. Two pathologists, blinded to outcome, independentlyread and adjudicated scores for all clinical specimens. A sample wasdefined as PD-L1-positive if 5% of tumor cells exhibited membrane PD-L1staining of any intensity in a section with 100 evaluable cells.

Statistical Analysis

All treated patients (N=86) as of February 2013 were used to describebaseline characteristics, safety, and absolute lymphocyte count (ALC),and analysis of PD-L1 staining. The efficacy population consisted of 82response-evaluable patients who received at least one dose of studytherapy, had measurable disease at baseline, and one of thefollowing: >1 on-treatment tumor evaluation, clinical progression, ordeath before the first on-treatment tumor evaluation. AEs were codedusing MedDRA, version 15.1. Select AEs with potential immunologicetiologies were identified using a pre-defined list of MedDRA terms.Best overall responses were derived programmatically fromtumor-measurements provided by the study-site radiologist andinvestigators per modified WHO (mWHO) or immune-related responsecriteria (Wolchok et al. 2009). Complete and partial responses wereconfirmed by at least one subsequent tumor assessment. An analysis alsowas performed to assess the magnitude of reduction in target lesions byradiographic assessment. A response was characterized as deep if areduction of 80% from baseline measurements was noted. Unconfirmedresponses as of the date of this analysis were also included in anestimate of aggregate clinical activity.

Example 16 Safety Evaluations on MEL Patients Treated with Anti-PD-1Plus Anti-CTLA-4

For the concurrent regimen (n=53), AEs of any grade, irrespective ofattribution, were observed in 98% of patients. Treatment-related AEswere observed in 93% of patients with the most common being rash (55%),pruritus (47%), fatigue (38%), and diarrhea (34%). Grade 3-4 AEs,irrespective of attribution, were observed in 72% of patients, whilegrade 3-4 treatment-related events were noted in 53%, with the mostcommon being elevations of lipase (13%), aspartate aminotransferase(13%) and alanine aminotransferase (11%). Six of 28 (21%) patients hadgrade 3-4 dose-limiting treatment-related events. Treatment-relatedserious AEs were reported in 49% of patients. Common grade 3-4treatment-related select AEs included hepatic (15%), gastrointestinal(9%), and renal (6%) events. Isolated cases of pneumonitis and uveitiswere seen, consistent with historical monotherapy experiences. Eleven(21%) patients discontinued due to treatment-related AEs.

Cohort 3 (3 mg/kg nivolumab+3 mg/kg ipilimumab) exceeded the MTD (3 of 6patients experienced asymptomatic grade 3-4 elevated lipase thatpersisted for 3 weeks). Cohort 2 (1 mg/kg nivolumab+3 mg/kg ipilimumab)was identified as the MTD (grade 3 uveitis, grade 3 elevated AST/ALT in1 patient each).

For the sequenced regimen (n=33), AEs of any grade, irrespective ofattribution, were observed in 29 (88%) patients. Treatment-related AEswere observed in 24 (73%) patients, with the most common includingpruritus (18%) and lipase elevation (12%). Grade 3-4 AEs, irrespectiveof attribution, were observed in 11 (33%) patients, while grade 3-4treatment-related AEs were observed in 6 (18%) patients, with lipaseelevation as the most common event (6%). Treatment-related serious AEswere reported in 7 (21%) patients. Grade 3-4 endocrine events were notedas treatment-related select AEs in 2 patients. One patient had grade-2pneumonitis. Three (9%) patients discontinued due to treatment-relatedAEs.

For both the concurrent and sequenced regimens, treatment-related AEswere manageable and generally reversible with immunosuppressants and/orreplacement therapy (for endocrinopathies) per previously algorithmspreviously established for ipilimumab (see YERVOY® package insert).Amongst the 86 patients treated on the study, 28 of 73 patients (38%)with drug-related adverse events required management with systemicglucocorticoids. Three patients required additional immunosuppressivetherapy with infliximab (2 patients) or mycophenolate mofetil (1patient). No treatment-related deaths were reported. Additional detailsof AEs and their management are provided in (Wolchok et al. 2013a).

Example 17 Efficacy Exhibited by Combination of Anti-PD-1 andAnti-CTLA-4 in MEL Patients

Clinical activity was observed with both the concurrent and sequencedregimens (Tables 10 and 11). In the concurrent-regimen cohorts,confirmed objective responses (OR) by mWHO criteria were observed in 21of 52 (40%; 95% CI: 27-55) response-evaluable patients across all doses.After noting several patients who demonstrated major responses(approaching CR), the number of patients with at least 80% tumorreduction, an empirical threshold chosen because it represents a levelof tumor regression that approaches complete response, was assessed.This depth of response was uncommon in published studies of checkpointblockade (Hodi et al., 2010; Topalian et al., 2012b). Sixteen patientshad ≧80% tumor reduction at 12 weeks, including 5 CRs (Table 10, FIGS.14A and 15-17). In addition to the 21 patients with OR by mWHO criteria,4 patients experienced an objective response by immune-related responsecriteria and 2 patients had unconfirmed responses. These patients werenot included in the calculation of ORRs. For the concurrent regimen,overall evidence of clinical activity (conventional, unconfirmed, orimmune-related response or SD ≧24 weeks) was observed in 65% (95% CI:51-78; Table 10) of patients. The profound impact of the concurrentcombination can best be appreciated in the waterfall plot (FIG. 14B).Responses were ongoing among 19 of 21 responders, with durations rangingfrom 6.1+ to 72.1+ weeks at the time of data analysis (Table 12). Forpatients treated at the MTD (cohort 2, 1 mg/kg nivolumab+3 mg/kgipilimumab), OR occurred in 9 of 17 (53%; 95% CI: 28-77) patients,including 3 CRs. All 9 responders achieved ≧80% tumor reduction at theirfirst scheduled on-treatment assessment (Table 10 and FIG. 14A).

For patients in the sequenced-regimen cohorts, 6 of 30 patients achievedOR (20%; 95% CI: 8-39) including 1 CR. Four (13%) patients achieved 80%tumor reduction at 8 weeks (Table 11 and FIG. 18). Additional patientshad immune-related (n=3) or unconfirmed (n=3) responses. When objective,immune-related or unconfirmed responses or SD ≧24 weeks are considered,evidence of clinical activity for the sequenced regimen was observed in43% (95% CI: 26-63). The waterfall plot reveals that patients who didnot respond to prior ipilimumab can respond to subsequent nivolumab(FIG. 18C).

TABLE 10 Clinical Activity of Patients who Received the ConcurrentRegimen of Nivolumab and Ipilimumab* 80% Response- Objective AggregateTumor Evaluable Response Clinical Reduction Patients^(†) CR PR uPR^(‡)irPR^(§) SD ≧ 24 irSD^(§) ≧ 24 Rate^(¶) % Activity Rate^(∥) % at 12 wkCohort Dose n n n n n wk n wk n [95% CI] [95% CI] n (%) 1 0.3 mg/kgnivolumab + 14 1 2 0 2 2 (14) 0 21 [5-51]  50 [23-77] 4 (29) 3 mg/kgipilimumab 2 1 mg/kg nivolumab + 17 3 6 0 0 0 2 (12) 53 [28-77] 65[38-86]  7 (41)** 3 mg/kg ipilimumab 2a 3 mg/kg nivolumab + 15 1 5 2 1 2(13) 0 40 [16-68] 73 [45-92] 5 (33) 1 mg/kg ipilimumab 3 3 mg/kgnivolumab + 6 0 3 0 1 0 1 (17) 50 [12-88]  83 [36-100] 0 3 mg/kgipilimumab All Concurrent treatment 52 5 16 2 4 4 (8)  3 (6)  40 [27-55]65 [51-78] 16 (31)  *CR denotes complete response, PR partial response,uPR unconfirmed partial response, irPR immune-related partial response,SD stable disease, irSD immune-related stable disease.^(†)Response-evaluable patients were those who received at least onedose of study therapy, had measurable disease at baseline, and one ofthe following: 1) at least one on-treatment tumor evaluation, 2)clinical progression, or 3) death prior to the first on-treatment tumorevaluation. ^(‡)Patients who had a PR after one tumor assessment but didnot have sufficient follow-up time for confirmation of the initial PR.^(§)Patients who had target tumor-lesion reduction in the presence ofnew lesions, consistent with immune-related PR or SD. ^(¶)[(CR + PR)/no.response-evaluable patients] × 100. Confidence intervals were estimatedby the Clopper-Pearson method. ^(∥)[(CR + PR + uCR + uPR + irPR + SD ≧24 wk + irSD ≧ 24 wk)/no. response-evaluable patients] × 100. **Twoadditional patients in cohort 2 achieved ≧80% tumor reduction at theirfirst scheduled assessment, which was conducted after week 12.

TABLE 11 Clinical Activity of Patients who Received the SequencedRegimen of Nivolumab and Ipilimumab* Aggregate 80% Response- ObjectiveClinical Tumor Evaluable Response Activity Reduction Patients^(†) CR PRuPR^(‡) irPR^(§) SD ≧ 24 irSD^(§) ≧ 24 Rate^(¶) % Rate^(∥) % at 8 wkCohort Dose n n n n n wk n wk n [95% CI] [95% CI] n (%) 6 1 mg/kgnivolumab + 16 1 5 2 2 1 (6) 0 38 [15-65] 69 [41-89] 4 (25) prioripilimumab 7 3 mg/kg nivolumab + 14 0 0 1 1 0 0 0 14 [2-43]  0 prioripilimumab All Sequenced treatment 30 1 5 3 3 1 (3) 0 20 [8-39]  43[26-63] 4 (13) *CR denotes complete response, PR partial response, uPRunconfirmed partial response, irPR immune-related partial response, SDstable disease, irSD immune-related stable disease.^(†)Response-evaluable patients were those who received at least onedose of study therapy, had measurable disease at baseline, and one ofthe following: 1) at least one on-treatment tumor evaluation, 2)clinical progression, or 3) death prior to the first on-treatment tumorevaluation. ^(‡)Patients who had a PR after one tumor assessment but didnot have sufficient follow-up time for confirmation of the initial PR.

TABLE 12 Duration of Confirmed Objective Responses of IndividualPatients Cohort Dose Response Duration of Response, weeks 1 0.3 mg/kgnivolumab + 3 mg/kg ipilimumab CR 48.0+ PR 72.1+, 65.4+ 2 1 mg/kgnivolumab + 3 mg/kg ipilimumab CR 36.1+, 28.9+, 24.7+ PR 54.4+, 52.6,23+, 18.4+, 14.0+, 11.1+ 2a 3 mg/kg nivolumab + 1 mg/kg ipilimumab CR7.7+ PR 18.1+, 12.3, 6.6+, 6.4+, 6.1+ 3 3 mg/kg nivolumab + 3 mg/kgipilimumab PR 38.7+, 31.3+, 12.1+ 6 1 mg/kg nivolumab + prior ipilimumabCR 24.1+ PR 16.1+, 12.1+, 8.7+, 8.4+, 8.1+ 7 3 mg/kg nivolumab + prioripilimumab CR NA* PR NA* *Not available; as of the date of thisanalysis, no confirmed objective responses had been reported.

Assessment of Tumor PD-L1 Expression and Absolute Lymphocyte Count

Tumor PD-L1 expression and alterations in the peripheral blood ALC havebeen explored as biomarkers for nivolumab and ipilimumab monotherapy,respectively (Topalian et al., 2012b; Berman et al., 2009; Ku et al.,2010; Postow et al., 2012; Delyon et al., 2013). Tumor PD-L1 expressionwas characterized via IHC staining and pharmacodynamic changes inperipheral blood ALC was analyzed. Using a ≧5% cut off to define PD-L1positivity, tumor specimens from 21 of 56 (38%) patients werePD-L1-positive. ORs were seen in patients with either PD-L1-positive(6/13) or PD-L1-negative (9/22) tumors amongst patients treated with theconcurrent regimen (post-hoc P-value >0.99; Fisher's exact test). Insequenced-regimen cohorts, a numerically higher number of overallresponses were seen in patients with PD-L1-positive tumor samples (4/8)compared with patients with PD-L1-negative tumors (1/13), but thenumbers are small.

In contrast to observations with ipilimumab monotherapy, a consistentrise in ALC from baseline was not detected in patients treated with theconcurrent combination or in patients treated with nivolumab followingipilimumab therapy. In the concurrent-regimen cohorts, patients with alow ALC at weeks 5 to 7 (<1000 cells/μL) (Ku et al., 2010) had similarOR (43%) compared with patients with a normal ALC at weeks 5 to 7 (40%).Likewise, in the sequenced-regimen cohorts, 17% of patients with low ALChad OR and 23% of patients with normal or high ALC had OR.

Sequence Listing Summary SEQ ID NO: Description 1 V_(H) amino acidsequence of 17D8 2 V_(H) amino acid sequence of 2D3 3 V_(H) amino acidsequence of 4H1 4 V_(H) amino acid sequence of 5C4 5 V_(H) amino acidsequence of 4A11 6 V_(H) amino acid sequence of 7D3 7 V_(H) amino acidsequence of 5F4 8 V_(L) amino acid sequence of 17D8 9 V_(L) amino acidsequence of 2D3 10 V_(L) amino acid sequence of 4H1 11 V_(L) amino acidsequence of 5C4 12 V_(L) amino acid sequence of 4A11 13 V_(L) amino acidsequence of 7D3 14 V_(L) amino acid sequence of 5F4 15 V_(H) amino acidsequence of 3G10 16 V_(H) amino acid sequence of 12A4 17 V_(H) aminoacid sequence of 10A5 18 V_(H) amino acid sequence of 5F8 19 V_(H) aminoacid sequence of 10H10 20 V_(H) amino acid sequence of 1B12 21 V_(H)amino acid sequence of 7H1 22 V_(H) amino acid sequence of 11E6 23 V_(H)amino acid sequence of 12B7 24 V_(H) amino acid sequence of 13G4 25V_(L) amino acid sequence of 3G10 26 V_(L) amino acid sequence of 12A427 V_(L) amino acid sequence of 10A5 28 V_(L) amino acid sequence of 5F829 V_(L) amino acid sequence of 10H10 30 V_(L) amino acid sequence of1B12 31 V_(L) amino acid sequence of 7H1 32 V_(L) amino acid sequence of11E6 33 V_(L) amino acid sequence of 12B7 34 V_(L) amino acid sequenceof 13G4 35 V_(H) amino acid sequence of 28-8 36 V_(L) amino acidsequence of 28-8 37 Heavy chain CDR1 sequence of 28-8 38 Heavy chainCDR2 sequence of 28-8 39 Heavy chain CDR3 sequence of 28-8 40 Lightchain CDR1 sequence of 28-8 41 Light chain CDR2 sequence of 28-8 42Light chain CDR1 sequence of 28-8

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1. A method of treating a subject afflicted with a cancer comprisingadministering to the subject a therapeutically effective amount of anantibody or an antigen-binding portion thereof that disrupts theinteraction between Programmed Death-1 (PD-1) and Programmed DeathLigand-1 (PD-L1), wherein the antibody or antigen-binding portionthereof binds specifically to PD-1 or to PD-11.
 2. The method of claim1, wherein the cancer is selected from the group consisting of melanoma,renal cell carcinoma, squamous non-small cell lung cancer (NSCLC),non-squamous NSCLC, colorectal cancer, castration-resistant prostatecancer, ovarian cancer, gastric cancer, hepatocellular carcinoma,pancreatic carcinoma, squamous cell carcinoma of the head and neck,carcinomas of the esophagus, gastrointestinal tract and breast, and ahematological malignancy.
 3. The method of claim 1, wherein thetherapeutically effective amount of the antibody or antigen-bindingportion thereof comprises a dose ranging from 0.1 to 10.0 mg/kg bodyweight which is administered at a dosing schedule of once per week, onceevery two weeks, or once a month.
 4. A method for immunotherapy of asubject afflicted with cancer, which method comprises: (a) selecting asubject that is a suitable candidate for immunotherapy, the selectingcomprising: (i) providing a test tissue sample obtained from a patientwith cancer of the tissue, the test tissue sample comprising tumor cellsand tumor-infiltrating inflammatory cells; (ii) assessing the proportionof cells in the test tissue sample that express PD-L1 on the cellsurface; and (iii) selecting the subject as a suitable candidate basedon an assessment that the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface exceeds a predetermined thresholdlevel; and (b) administering a composition comprising a therapeuticallyeffective amount of an anti-PD-1 antibody to the selected subject. 5.The method of claim 4, wherein the proportion of cells that expressPD-L1 is assessed by performing an assay to determine the presence ofPD-L1 polypeptide on the surface of cells in the test tissue sample. 6.The method of claim 5, wherein the test tissue sample is aformalin-fixed paraffin-embedded (FFPE) tissue sample.
 7. The method ofclaim 6, wherein the presence of PD-L1 polypeptide is determined usingan automated IHC assay.
 8. The method of claim 7, wherein the IHC assayis performed using an anti-PD-L1 monoclonal antibody to bind to thePD-L1 polypeptide, wherein the anti-PD-L monoclonal antibody is selectedfrom 28-8, 28-1, 28-12, 29-8, and 5H1.
 9. The method of claim 4, whereinthe predetermined threshold level is 1% of tumor cells or a singletumor-infiltrating inflammatory cell expressing cell surface PD-L1 asdetermined by automated IHC using mAb 28-8.
 10. A method for treatmentof a subject afflicted with cancer, which method comprises: (a)selecting a subject that is not suitable for anti-PD-1 antibodyimmunotherapy, the selecting comprising: (i) providing a test tissuesample obtained from a patient with cancer of the tissue, the testtissue sample comprising tumor cells and tumor-infiltrating inflammatorycells; (ii) assessing the proportion of cells in the test tissue samplethat express PD-L1 on the cell surface; and (iii) selecting the subjectas not suitable for anti-PD-1 antibody immunotherapy based on anassessment that the proportion of cells in the test tissue sample thatexpress PD-L1 on the cell surface is less than a predetermined thresholdlevel; and (b) administering a standard-of-care therapeutic other thanan anti-PD-1 antibody to the selected subject.
 11. The method of claim10, wherein the proportion of cells in the test tissue sample thatexpress PD-L1 on the cell surface is determined with a monoclonalantibody or an antigen-binding portion thereof that binds specificallyto a cell surface-expressed PD-L1 polypeptide in a formalin-fixed,paraffin-embedded (FFPE) tissue sample.
 12. The method of claim 11,wherein the monoclonal antibody or antigen binding portion thereofcomprises the CDR1, CDR2 and CDR3 regions in a heavy chain variableregion comprising consecutively linked amino acids having the sequenceset forth in SEQ ID NO: 35, and the CDR1, CDR2 and CDR3 regions in alight chain variable region comprising consecutively linked amino acidshaving the sequence set forth in SEQ ID NO:
 36. 13. The method of claim11, wherein the monoclonal antibody or antigen binding portion thereofcomprises a heavy chain variable region comprising consecutively linkedamino acids having the sequence set forth in SEQ ID NO: 35, and a lightchain variable region comprising consecutively linked amino acids havingthe sequence set forth in SEQ ID NO:
 36. 14. A method of treating asubject afflicted with a cancer comprising administering to the subject:(a) an antibody or an antigen-binding portion thereof that specificallybinds to and inhibits Programmed Death-1 (PD-1); and (b) an antibody oran antigen-binding portion thereof that specifically binds to andinhibits Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4); each antibody beingadministered at a dosage ranging from 0.1 to 20.0 mg/kg body weight in aconcurrent regimen comprising: (i) an induction dosing schedulecomprising combined administration of the anti-PD-1 and anti-CTLA-4antibodies at a dosing frequency of at least once every 2, 3 or 4 weeks,or at least once a month, for at least 2, 4, 6, 8 or 10 doses, followedby administration of the anti-PD-1 alone at a dosing frequency of atleast once every 2, 3 or 4 weeks, or at least once a month, for at least2, 4, 6, 8 or 12 doses; followed by (ii) a maintenance dosing schedulecomprising combined administration of the anti-PD-1 and anti-CTLA-4antibodies at a dosing frequency of at least once every 8, 12 or 16weeks, or at least once a quarter, for at least 4, 6, 8, 10, 12 or 16doses.
 15. The method of claim 14, wherein the anti-PD-1 and anti-CTLA-4antibodies are administered at the following dosages: (a) 0.1 mg/kganti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody; (b) 0.3 mg/kganti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody; (c) 1 mg/kganti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody; (d) 3 mg/kganti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody; (e) 5 mg/kganti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody; (f) 10 mg/kganti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody; (g) 0.1 mg/kganti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody; (h) 0.3 mg/kganti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody; (i) 1 mg/kganti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody; (j) 3 mg/kganti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody; (k) 5 mg/kganti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody; or (l) 10 mg/kganti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody.
 16. A method oftreating a subject afflicted with a cancer, the subject havingpreviously been treated with an anti-CTLA-4 antibody, which methodcomprises administering to the subject in a sequenced regimen anantibody or an antigen-binding portion thereof that specifically bindsto and inhibits PD-1 at a dosage ranging from 0.1 to 20.0 mg/kg bodyweight and at a dosing frequency of at least once every week, at leastonce every 2, 3 or 4 weeks, or at least once a month, for up to 6 to upto 72 doses.
 17. The method of claim 16, wherein the anti-PD-1 antibodyis nivolumab.
 18. The method of 16, wherein the anti-CTLA-4 antibody isipilimumab.
 19. (canceled)
 20. A kit for treating a subject afflictedwith a cancer, the kit comprising: (a) a dosage ranging from 0.1 to 20.0mg/kg body weight of an antibody or an antigen-binding portion thereofthat specifically binds to and inhibits PD-1; (b) a dosage ranging from0.1 to 20.0 mg/kg body weight of an antibody or an antigen-bindingportion thereof that specifically binds to and inhibits CTLA-4; and (c)instructions for using the anti-PD-1 and anti-CTLA-4 antibodies theconcurrent regimen method of claim
 14. 21. A kit for treating a subjectafflicted with a cancer, the kit comprising: (a) a dosage ranging from0.1 to 20.0 mg/kg body weight of an antibody or an antigen-bindingportion thereof that specifically binds to and inhibits PD-1; and (b)instructions for using the anti-PD-1 antibody in the sequenced regimenmethod of claim
 16. 22. The method of claim 15, wherein the anti-PD-1antibody is nivolumab.
 23. The method of claim 15, wherein theanti-CTLA-4 antibody is ipilimumab.